Dose escalation enzyme replacement therapy for treating acid sphingomyelinase deficiency

ABSTRACT

The invention relates to dose escalation enzyme replacement therapy using acid sphingomyelinase (ASM) for the treatment of human subjects having acid sphingomyelinase deficiency (ASMD), and, in particular, patients with non-neurological manifestations of Niemann-Pick Disease (NPD), and in certain embodiments, NPD type B.

This application is a continuation of U.S. patent application Ser. No.16/209,336, filed Dec. 4, 2018, now abandoned, which is a continuationof U.S. patent application Ser. No. 15/485,604, filed Apr. 12, 2017, nowU.S. Pat. No. 10,188,705, which is a continuation of U.S. patentapplication Ser. No. 14/793,838, filed Jul. 8, 2015, now U.S. Pat. No.9,655,954, which is a continuation of U.S. patent application Ser. No.14/156,894, filed Jan. 16, 2014, now U.S. Pat. No. 9,114,139, which is acontinuation of U.S. patent application Ser. No. 13/679,623, filed Nov.16, 2012, now U.S. Pat. No. 8,709,408, which is a divisional of U.S.patent application Ser. No. 12/870,790, filed Aug. 28, 2010, now U.S.Pat. No. 8,349,319, which claims the benefit of U.S. Provisional PatentApplication No. 61/238,113, filed Aug. 28, 2009, each of which isincorporated herein by reference in its entirety.

SEQUENCE LISTING

This application incorporates by reference in its entirety the ComputerReadable Form (CRF) of a Sequence Listing in ASCII text format submittedvia EFS-Web. The Sequence Listing text file submitted via EFS-Web,entitled 06923-331-999 SEQ LISTING.txt, was created on May 25, 2020, andis 6,436 bytes in size.

1. INTRODUCTION

The invention relates to dose escalation enzyme replacement therapyusing acid sphingomyelinase (ASM) for the treatment of human subjectshaving acid sphingomyelinase deficiency (ASMD), and, in particular, thenon-neurological manifestations of Niemann Pick Disease (NPD), and incertain embodiments, NPD type B.

2. BACKGROUND

Acid sphingomyelinase, E.C. 3.1.4.12, (ASM) is a lysosomalphosphodiesterase enzyme that hydrolyzes sphingomyelin, a phospholipidstorage substance found in the brain, liver, lungs, spleen and lymphnodes, to ceramide and phosphorylcholine. Deficiencies in ASM activityresult in the inability of the body to break down sphingomyelin, causinga form of the lysosomal storage disease termed Niemann-Pick disease.

Niemann-Pick disease is an inherited autosomal recessive lipid storagedisorder characterized by excessive accumulation of sphingomyelin in thelysosomes of cells such as macrophages and neurons, which impairs normalcellular function. Niemann-Pick Type A is a rapidly progressiveneurodegenerative disease in infants and typically results in deathwithin two to three years of age. Niemann-Pick Type B results in theenlargement of the liver and spleen, and respiratory distress with deathgenerally ensuing by early adulthood. These two forms of Niemann-Pickdisease which are both associated with ASM deficiencies are referred tocollectively herein as Niemann-Pick disease, or ASM deficiency (ASMD).Other types of Niemann-Pick disease, e.g., Type C, do not involvemutations in the ASM gene and are not directly attributable to thefunction of ASM. The nature of the biochemical and molecular defectsthat underlie the remarkable clinical heterogeneity of the A and Bsubtypes remains unknown. Although patients with both subtypes haveresidual ASM activity (about 1 to 10% of normal), biochemical analysiscannot reliably distinguish the two phenotypes. Moreover, the clinicalcourse of Type B NPD is highly variable, and it is not presentlypossible to correlate disease severity with the level of residual ASMactivity.

NPD occurs more frequently among individuals of Ashkenazi Jewishancestry than in the general population. It is estimated that theincidence of the type A disease among Ashkenazi Jews is about 1 in40,000, a gene frequency (q) of about 1 in 200, and a heterozygotecarrier frequency (2 pq) of 1 in 100 (Goodman, 1979, in “GeneticDisorders Among The Jewish People”, John Hopkins Univ. Press, Baltimore,pp. 96-100). The heterozygote carrier incidence of type B NPD in theAshkenazi Jewish population is less frequent (Goodman, supra). Thecombined heterozygote carrier frequency for types A and B NPD has beenestimated to be about 1 in 70 among individuals of Ashkenazi Jewishdecent. Although the enzymatic diagnosis of affected patients witheither type A or B NPD can be made reliably (Spence and Callahan,supra), the enzymatic detection of obligate heterozygotes has provenproblematic, particularly using peripheral leukocytes as the enzymesource. Presumably, the occurrence of neutral sphingomyelinases in somesources and/or the presence of residual ASM activity resulting from themutant allele have contributed to the inability to reliably discriminatecarriers for either disease subtype. Even the use of cultured skinfibroblasts, which do not express the neutral sphingomyelinase, has notprovided unambiguous results with heterozygotes. In epidemiologicstudies conducted in individual countries, the combined incidence ofNiemann-Pick A and B disease in several countries in the world isestimated to range from 1 in 167,000 to 1 in 250,000 newborns (Miekle etal., 1999 JAMA 281(3):249-254; Poorthuis et al., 1999 Hum Genet105:151-156; Pinto et al., 2004 Euro. J. Hum. Gene. 12:87-92). Theheterozygote carrier rate is believed to range from 1 in 200 to 1 in 250individuals.

Enzyme replacement therapy has been used for other lysosomal storagediseases. Enzyme replacement therapy attempts to supplement thedeficient enzyme activity with exogenously supplied enzyme. In the caseof enzyme replacement therapy for Niemann-Pick disease, the goal wouldbe to enable the affected individual to process sphingomyelin and avoidits buildup within the lysosomes. To be effective, such therapyinitially would require a sufficiently large amount of the replacementenzyme to break down the accumulated sphingomyelin as well as continuedadministration of replacement enzyme to avoid further accumulation ofsphingomyelin.

3. SUMMARY

The invention relates to dose escalation enzyme replacement therapy forthe treatment of human subjects having ASMD—particularly subjects havingnon-neurological manifestations of NPD, and in particular embodiments,NPD type B. More particularly, the enzyme, ASM, is administered to suchpatients at an initial low, non-toxic dose that is then escalated insubsequent administrations. The highest dose of ASM tolerated by thepatient can then be used as a maintenance dose. Alternatively, atherapeutically effective dose less than the highest dose tolerated canbe used as a maintenance dose.

The invention is based, in part, on the discovery that doses of ASM thatwould be required to clear accumulated sphingomyelin substrate in humansubjects, i.e., ASMD patients or Niemann-Pick patients, result in toxicside effects (including clinical signs of toxicity). This is especiallysurprising in the less severe form of ASMD, NPD type B patients who aredeficient but have at least some enzyme activity.

More particularly, treatment of NPD would require doses high enough toachieve adequate distribution of the ASM enzyme in organs of pathology(e.g., in particular, the liver, spleen, lungs, heart, kidney andbrain). Studies in an ASM knockout mouse model (ASKMO mice) showed thatthe majority of recombinant human ASM (rhASM) administered distributesto the liver and spleen where it reduces substrate, but to a much lesserextent in lung, heart and brain (Miranda et al. FASEB 2000, 14:1988; seealso, FIG. 9B of He et al., 1999, Biochimica et Biophsyica Acta 1432:251-264). In subsequent studies using higher doses of rhASM in the ASMKOmouse model, substrate was reduced and toxicity was not observed atdoses ≤3.0 mg/kg; in fact, clinical symptoms of toxicity was notobserved until doses ≥10 mg/kg were used. See, “Dose ResponsiveToxicological Findings Following Intravenous Administration ofRecombinant Human Acid Sphingomyelinase (rhASM) to Acid SphingomyelinaseKnock-out (ASMKO) Mice. C. Nickerson, J. Murray, A. Vitsky, M. Hawes, S.Ryan, P. Ewing, B. Thurberg, L. Andrews. Dept Pharm/Tox, Pathology,Genzyme Corp., Framingham, Mass., American Society of Human Genetics2005; and Elevations of Pro-Inflammatory Cytokines and Decreases inCardiovascular Hemodynamics Following Intravenous Administration ofRecombinant Human Acid Sphingomyelinase (rhASM) to Acid SphingomyelinaseKnock-out (ASMKO) Mice. J. Murray, A. M. D'Angona, C. Nickerson, A.Vitsky, M. Hawes, S. Ryan, P. Ewing, B. Thurberg, L. Andrews. Dept.Pharmacology/Toxicology & Pathology, Genzyme Corp., Framingham, Mass.,Society of Toxicology 2006.

Based on these ASKMO data, we treated non-neuronopathic ASMD humansubjects with a conservative maximum dose of 1.0 mg/kg rhASM asdescribed in Section 6, infra. Quite unexpectedly, toxicity in the humansubjects, including the onset of related adverse events with clinicalsymptoms, was observed using doses as low as 0.3 mg/kg! This result wasespecially surprising, since the ASM enzyme is absent in the knock outmouse model which should reflect a more severe condition than in thesehuman subjects having at least some enzyme activity and relatively milddisease.

While not intended to be bound by any theory, the toxic side effectsthat occur with ASM treatment may result from breakdown of storedsphingomyelin substrate in the ASMD patient and release of the product,ceramide that is pro-apoptotic and induces a pro-inflammatory cytokineresponse and hyperbilirubinemia. To address this issue, we havedeveloped a regimen to allow for the safe administration of high dosesof the ASM enzyme required to achieve adequate distribution in organs ofpathology. In accordance with this regimen, initial treatment with ASMat very low doses is used to achieve a slow degradation of the storedsubstrate which is accompanied by fewer side effects. As the substrateis depleted in the subject (as the storage substrate is “debulked”), thedose can be escalated safely.

In accordance with this protocol, a low, non-toxic dose of the ASMenzyme is initially administered to a NPD disease patient and the doseis escalated over time. As the dose of the ASM enzyme is escalated, thepatient can be monitored for total bilirubin concentration, theproduction of acute phase reactants, the production of inflammatorymediators, and related adverse events. The administration of a low doseof ASM and the escalation of the dose facilitates the debulking of theaccumulated sphingomyelin. Once the patient is debulked, higher dosesmay be safely administered to the patient to ensure adequatedistribution of the ASM enzyme to target organs (e.g., liver, spleen,lungs, heart, kidney, brain, bone marrow, skeleton, joints, etc.). Incertain embodiments, the maximum dose tolerated by the patient can beused as the maintenance dose. In some embodiments, based upon apatient's condition, the maintenance dose may be increased or decreasedover time.

In certain embodiments, treatment of the patient is monitored bymeasuring the plasma sphingomyelin levels, plasma ceramide levels, theproduction of “acute phase reactants” and inflammatory mediators thatare a measure of inflammatory responses, bilirubin concentrations(total, direct, and indirect), and/or other biochemical markers toensure a stable response before elevating the dose to the next level.These markers include, but are not limited to C-reactive protein (CRP)or high sensitivity CRP (hs-CRP), cytokines (e.g., IL-8, IL-6),calcitonin and ferritin. In specific embodiments, the patient may bemonitored for one or more related adverse events, which may include, butare not limited to, constitutional symptoms (e.g., fever, nausea,vomiting, pain, myalgia) and jaundice.

Doses less than 1 mg/kg are preferable for initiating treatment. Theinitial dose is successively elevated until a therapeutic dose isachieved. Such dose escalation can be used to determine the highesttolerated dose. For example, once the patient is debulked of theaccumulated sphingomyelinase substrate, the dose may be furtherescalated until toxicity is observed. The maintenance dose can beadjusted accordingly, and can be continually and periodically readjusteddepending on the status of the patient.

In a specific embodiment, a method for treating a human subject havingan acid sphingomyelinase deficiency, comprises: (a) a regimen fordebulking accumulated sphingomyelin substrate in the human subjectcomprising: (i) administering an initial low non-toxic dose of ASM tothe human subject; (ii) administering successively higher doses of ASMto the human subject, and monitoring the subject for one or more adverseside effects after each successive dose as indicated by elevatedbilirubin or a related adverse event; and (b) a maintenance regimencomprising administering a dose equal to or less than the highest dosetolerated by the subject as the maintenance dose for the subject.

In another specific embodiment, a method for treating a human subjecthaving an acid sphingomyelinase deficiency, comprises administeringrhASM in an escalating dose regimen at the following sequential doses:0.1 mg/kg; 0.3 mg/kg; 0.6 mg/kg; and 1.0 mg/kg, wherein each dose ofrhASM is administered at least twice, and each dose is administered attwo week intervals, and wherein the patient is monitored for toxic sideeffects before elevating the dose to the next level.

In another specific embodiment, described herein is an acidsphingomyelinase (ASM) for use in the treatment of an acidsphingomyelinase deficiency in a human subject prepared to beadministered: (a) in a regimen for debulking accumulated sphingomyelinsubstrate comprising: (i) administration of an initial low non-toxicdose of acid sphingomyelinase (ASM); (ii) administration of successivelyhigher doses of ASM, and monitoring the subject for one or more adverseside effects after each successive dose as indicated by elevatedbilirubin or a related adverse event; and (b) in a maintenance regimencomprising administration of a dose equal to or less than the highestdose tolerated by the subject as the maintenance dose for the subject.

In another specific embodiment, described herein is a recombinant humanASM for use in the treatment of an acid sphingomyelinase deficiency in ahuman subject prepared to be administered in an escalating dose regimenat the following sequential doses: 0.1 mg/kg; 0.3 mg/kg; 0.6 mg/kg; and1.0 mg/kg, wherein each dose of is administered at least twice, and eachdose is administered at two week intervals, and wherein the subject ismonitored for toxic side effects before elevating the dose to the nextlevel.

3.1. Terminology

As used herein, the terms “about” and “approximately” are usedinterchangeably in the context of a given value to refer to a rangearound a given value, wherein the resulting value is substantially thesame as the expressly recited value. In a specific embodiment “about”means within 10%, 15%, 25% of a given value or range.

As used herein, the term “elderly human” refers to a human 65 years orolder.

As used herein, the term “human adult” refers to a human that is 18years or older.

As used herein, the term “human child” refers to a human that is 1 yearto 18 years old.

As used herein, the term “human infant” refers to a newborn to 1 yearold year human.

As used herein, the term “human toddler” refers to a human that is 1year to 3 years old.

As used herein, the term “adverse event” refers to “any untoward medicaloccurrence in a patient or clinical investigation subject administered apharmaceutical product” as defined in the Clinical Data InterchangeStandards Consortium Study Data Tabulation Model standard terminology v.3.1.1. A “related adverse event” is an adverse event that has a casualrelationship with treatment.

As used herein, the term “maintenance dose(s)” and the like refers to adosage administered to ASMD patients to maintain the desired therapeuticeffect. In specific embodiments, the maintenance dose(s) maintains one,two, three, four or more the following desired therapeutic effects: (i)a reduction in spleen volume as assessed by techniques known in the art,e.g., MRI; (ii) a reduction in liver sphingomyelin levels as assessed bytechniques known in the art, e.g., biochemical analysis and/orhistomorphometric analysis of liver samples; (iii) an increase inexercise capacity as assessed by techniques known in the art, e.g.,maximum workload by cycle erogmetry, including percent predicted maximumworkload, peak oxygen consumption and carbon dioxide production; (iv) anincrease in pulmonary function as assessed by techniques known in theart, e.g., techniques described in American Thoracic Society, 1991, Am.Rev. Respir. Dis. 144: 1202-1218, such as diffusing capacity (DLco),percent predicted forced vital capacity (FVC) as measured by, e.g.,spirometric techniques, forced expiratory volume within 1 second (FEV₁)as measured by, e.g., spirometric techniques, and total lung capacity;(v) a decrease in bronchial alveolar lavage (BAL) sphingomyelin; (vi) adecrease in liver volume as assessed by techniques known in the art,e.g., MM: (vii) an improvement in lung appearance as assessed bytechniques known in the art, e.g., high resolution computed tomography(CT) scan or chest X-ray; (viii) a decrease in sphinomyelinconcentration in the liver, skin, plasma and dried blood spot (DBS) asmeasured by, e.g., tandem mass spectrometry; (ix) a reduction or theamelioration of the severity of ASMD and/or a symptom associatedtherewith; (x) a reduction in the duration of a symptom associated withASMD; (xi) the prevention in the recurrence of a symptom associated withASMD; (xii) a reduction in hospitalization of a subject; (vi) areduction in hospitalization length; (xiii) an increase in the survivalof a subject; (xiv) a reduction in mortality; (xv) a decrease inhospitalization rate; (xvi) a reduction in the number of symptomsassociated with ASMD; (xvii) an increase in symptom-free survival ofASMD patients; (xviii) an improvement in neurological function (e.g.,psychomotor function, social responsiveness, etc.); (xix) an improvementin lung clearance as measured by, e.g., BAL cell count and profile; (xx)a decrease in serum levels of chitotriosidase; (xxi) a decrease in serumlevels of chemokine (c-c) motif ligand 18 (CCL18); (xxii) an improvementin lipid profile (e.g., HDL, LDL, cholesterol, triglycerides, and totalcholesterol:HDL ratio); and (xxiii) improved quality of life as assessedby, e.g., a questionnaire. In certain embodiments, the maintenance doseis the highest or maximum dose tolerated by a patient.

In some embodiments, the maintenance dose is a dose of between 0.5 mg/kgto 1.5 mg/kg, 0.75 mg/kg to 1.25 mg/kg, 1 mg/kg to 2.5 mg/kg, 1 mg/kg to2.75 mg/kg, 1.5 mg/kg to 2.5 mg/kg, 1.5 mg/kg to 2.75 mg/kg, 2 mg/kg to2.5 mg/kg, 2 mg/kg to 2.75 mg/kg, 2.5 mg/kg to 2.75 mg/kg, 2.5 mg/kg to3 mg/kg, 3 mg/kg to 4 mg/kg, 3 mg/kg to 5 mg/kg, 4 mg/kg to 5 mg/kg, 2mg/kg to 5 mg/kg or 5 mg/kg to 10 mg/kg of ASM. In certain embodiments,the maintenance dose is a dose of between 5 mg/kg to 15 mg/kg, 10 mg/kgto 15 mg/kg, 10 mg/kg to 20 mg/kg, 15 mg/kg to 20 mg/kg, 20 mg/kg to 30mg/kg, 25 mg/kg to 50 mg/kg, 30 mg/kg to 40 mg/kg, 30 mg/kg to 45 mg/kgor 40 mg/kg to 50 mg/kg of ASM. In some embodiments, the maintenancedose is 0.75 mg/kg, 0.80 mg/kg, 0.85 mg/kg, 0.90 mg/kg, 0.95 mg/kg, 1mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.25 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5mg/kg, 1.75 mg/kg, or 2 mg/kg of ASM. In certain embodiments, themaintenance dose is 2.5 mg/kg, 2.75 mg/kg, 3 mg/kg, 3.25 mg/kg, 3.5mg/kg, 3.75 mg/kg, 4 mg/kg, 4.25 mg/kg, 4.5 mg/kg, 4.75 mg/kg, 5 mg/kg,5.5 mg/kg, 6 mg/kg, 6.5 mg/kg, 7 mg/kg, 7.5 mg/kg, 8 mg/kg, 8.5 mg/kg, 9mg/kg, 9.5 mg/kg or 10 mg/kg of ASM. In some embodiments, themaintenance does is 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 20mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg or 50 mg/kg ofASM. In some embodiments, the maintenance dose is at least 1 mg/kg, atleast 2 mg/kg, at least 3 mg/kg, at least 4 mg/kg, at least 5 mg/kg, atleast 6 mg/kg, at least 7 mg/kg, at least 8 mg/kg of ASM with thehighest dose being 10 mg/kg of ASM. In certain embodiments, themaintenance dose is at least 10 mg/kg, at least 15 mg/kg, at least 20mg/kg, at least 25 mg/kg, at least 30 mg/kg, or at least 35 mg/kg of ASMwith the highest dose being 50 mg/kg.

As used herein, the term “non-toxic dose(s)” and the like refers to adosage administered to ASMD patients without resulting in one, two,three or all of the following: (i) a moderate or severe related adverseevent as defined by a clinical symptom that interferes with normal dailyfunctioning and requires additional monitoring, intervention, ortreatment, or, an abnormal laboratory value or procedural result ofclinical concern that requires further monitoring, treatment, orinvestigation. See, e.g., the Clinical Data Interchange StandardsConsortium Study Data Tabulation Model standard terminology v.3.1.1;(ii) a total bilirubin value of greater than 1.5 mg/dL, 1.75 mg/dL, 2.0mg/dL, 2.1 mg/dL, 2.2 mg/dL, 2.3 mg/dL, 2.4 mg/dL, 2.5 mg/dL, 2.6 mg/dL,2.7 mg/dL, 2.75 mg/dL, 2.8 mg/dL, 2.9 mg/dL, 3.0 mg/dL, 3.1 mg/dL, 3.2mg/dL, 3.3 mg/dL, 3.4 mg/dL, 3.5 mg/dL, 3.6 mg/dL, 3.7 mg/dL, 3.8 mg/dL,3.9 mg/dL or 4 mg/dL or in the range of 2.1 mg/dL to 2.5 mg/dL, 2.1mg/dL to 3.0 mg/dL, or 2.1 mg/dL to 4 mg/dL that lasts for greater than18 hours, 24 hours, 36 hours, 48 hours or 72 hours, 5 days, one week,two weeks or three weeks after administration of the dose of ASM; (iii)a plasma ceramide concentration of greater than 8.2 μg/mL, 8.3 μg/mL,8.4 μg/mL, 8.5 μg/mL, 8.75 μg/mL, 9 μg/mL, 9.5 μg/mL, 10 μg/mL, 11μg/mL, 12 μg/mL, 13 μg/mL, 14 μg/mL, 15 μg/mL, 16 μg/mL, 17 μg/mL, 18μg/mL, 19 μg/mL, 20 μg/mL, 25 μg/mL, 30 μg/mL, 35 μg/mL, 40 μg/mL, 45μg/mL, 50 μg/mL, 55 μg/mL, 60 μg/mL, 65 μg/mL, 70 μg/mL, 75 μg/mL, or 80μg/mL, or in the range of 8.2 μg/mL to 10 μg/mL, 8.5 μg/mL to 10 μg/mL,9 μg/mL to 12 μg/mL, 10 μg/mL to 12 μg/mL, 10 μg/mL to 15 μg/mL, 10μg/mL to 20 μg/mL, 15 μg/mL to 20 μg/mL, or 20 μg/mL to 30 μg/mL 6hours, 8 hours, 10 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36hours, 48 hours or 72 hours after administration of the dose of ASM; or(iv) an acute phase response/reaction. The “non-toxic dose” of ASM mayvary depending upon, e.g., the stability of the enzyme used, theactivity of the enzyme used, and/or the route of administration of theenzyme. For example, the dose of a modified ASM enzyme with increasedactivity may be lower than the dosage of an unmodified ASM. One skilledin the art would be able to adjust the dose of enzyme administered basedon the stability of the enzyme, the activity of the enzyme, and/or theroute of administration of the enzyme.

An acute phase reaction is an early reaction (generally, e.g., within 12to 72 hours) following administration of ASM that is indicative of aninflammatory response. An acute phase response can be assessed by achange in the concentration of an acute phase reactant (such as, e.g.,CRP/hs-CRP, ferritin, fibrinogen, iron or transferrin), a change in thepercentage of neutrophils, a change in prothrombin time, or a change inpartial thromboplastin time. In a specific embodiment, an increase inCRP/hs-CRP concentration 6 hours, 8 hours, 12 hours, 18 hours, 24 hours,36 hours, 48 hours or 72 hours after administration of a dose of ASMrelative to a patient's CRP/hs-CRP concentration prior to administrationof ASM can be utilized as a measurement of an acute phase response. Inanother specific embodiment, a plasma CRP/hs-CRP concentration that isgreater than the normal plasma CRP/hs-CRP concentration 6 hours, 8hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours or 72hours after administration of the dose of ASM can be utilized as ameasurement of an acute phase response. In certain embodiments, a plasmaCRP/hs-CRP concentration of greater than approximately 8.1 mg/L, 8.2mg/L, 8.3 mg/L, 8.4 mg/L, 8.5 mg/L, 9 mg/L, 9.5 mg/L, 10 mg/L, 11 mg/L,or 12 mg/L, or in the range of 8.5 mg/L to 10 mg/L, or 8.5 mg/dL to 12mg/L, or 10 mg/L to 12 mg/L 6 hours, 8 hours, 12 hours, 16 hours, 18hours, 24 hours, 36 hours, 48 hours or 72 hours after administration ofthe dose of ASM can be used as a measurement of an acute phase response.

In a specific embodiment, an increase in ferritin concentration 6 hours,8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours or72 hours after administration of a dose of ASM relative to a patient'sferritin concentration prior to administration of ASM can be used as ameasurement of an acute phase response. In another specific embodiment,a plasma ferritin concentration that is greater than the normal plasmaferritin concentration 6 hours, 8 hours, 12 hours, 18 hours, 24 hours,36 hours, 48 hours or 72 hours after administration of the dose of ASMcan be used as a measurement of an acute phase response. In certainembodiments, a plasma ferritin concentration of greater thanapproximately 300 ng/mL, 325 ng/mL, 350 ng/mL, 375 ng/mL, 400 ng/mL, 425ng/mL, 450 ng/mL, 475 ng/mL, 500 ng/mL, 525 ng/mL, 550 ng/mL, 575 ng/mL,600 ng/mL, 625 ng/mL, 650 ng/mL, 675 ng/mL, 700 ng/mL, 725 ng/mL, 750ng/mL, 775 ng/mL, 800 ng/mL, 850 ng/mL, 900 ng/mL, 950 ng/mL, 1000ng/mL, 1050 ng/mL, 1100 ng/mL, 1150 ng/mL, or 1200 ng/mL or in the rangeof 600 ng/mL to 800 ng/mL, 650 ng/mL to 850 ng/mL, 600 ng/mL to 1000ng/mL, 600 ng/mL to 1200 ng/mL, 800 ng/mL to 1000 ng/mL, 900 ng/mL to1000 ng/mL, or 1000 ng/mL to 1200 ng/mL 6 hours, 8 hours, 12 hours, 16hours, 18 hours, 24 hours, 36 hours, 48 hours or 72 hours afteradministration of the dose of ASM can be used as a measurement of anacute phase response.

In a specific embodiment, an increase in plasma or serum IL-8concentration 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours,36 hours, 48 hours or 72 hours after administration of a dose of ASMrelative to a patient's IL-8 concentration prior to administration ofASM can be used as a measurement of an acute phase response. In anotherspecific embodiment, a plasma or serum IL-8 concentration that isgreater than the normal plasma IL-8 concentration 6 hours, 8 hours, 12hours, 18 hours, 24 hours, 36 hours, 48 hours or 72 hours afteradministration of the dose of ASM can be used as a measurement of anacute phase response. In certain embodiments, a plasma IL-8concentration of greater than approximately 24 pg/mL, 50 pg/mL, 75pg/mL, 100 pg/mL, 200 pg/mL, 300 pg/mL, 400 pg/mL, 500 pg/mL, 600 pg/mL,700 pg/mL, 800 pg/mL, or 900 pg/mL, 6 hours, 8 hours, 12 hours, 16hours, 18 hours, 24 hours, 36 hours, 48 hours or 72 hours afteradministration of the dose of ASM can be used as a measurement of anacute phase response.

In a specific embodiment, an increase in plasma or serum IL-6concentration 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours,36 hours, 48 hours or 72 hours after administration of a dose of ASMrelative to a patient's IL-6 concentration prior to administration ofASM can be used as a measurement of an acute phase response. In anotherspecific embodiment, a plasma or serum IL-6 concentration that isgreater than the normal plasma or serum IL-6 concentration 6 hours, 8hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours or 72 hoursafter administration of the dose of ASM can be used as a measurement ofan acute phase response. In certain embodiments, a plasma IL-6concentration of greater than approximately 4.4 pg/mL, 6 pg/mL, 8 pg/mL,10 pg/mL, 15 pg/mL, 20 pg/mL, 25 pg/mL, or 30 pg/mL, 6 hours, 8 hours,12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours or 72 hoursafter administration of the dose of ASM can be used as a measurement ofan acute phase response.

In a specific embodiment, an increase in plasma or serum calcitoninconcentration 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours,36 hours, 48 hours or 72 hours after administration of a dose of ASMrelative to a patient's calcitonin concentration prior to administrationof ASM can be used as a measurement of an acute phase response. Inanother specific embodiment, a plasma or serum calcitonin concentrationthat is greater than the normal plasma calcitonin concentration 6 hours,8 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours or 72 hoursafter administration of the dose of ASM can be used as a measurement ofan acute phase response. In certain embodiments, a plasma calcitoninconcentration of greater than approximately 9.4 pg/mL, 20 pg/mL, 30pg/mL, 40 pg/mL, 50 pg/mL, 75 pg/mL, 100 pg/mL, 150 pg/mL, 200 pg/mL, or250 pg/mL, 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36hours, 48 hours or 72 hours after administration of the dose of ASM canbe used as a measurement of an acute phase response.

In a specific embodiment, an increase in fibrinogen concentration 6hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48hours or 72 hours after administration of a dose of ASM relative to apatient's fibrinogen concentration prior to administration of ASM can beused as a measurement of an acute phase response. In another specificembodiment, a plasma fibrinogen concentration that is greater than thenormal plasma fibrinogen concentration 6 hours, 8 hours, 12 hours, 16hours, 18 hours, 24 hours, 36 hours, 48 hours or 72 hours afteradministration of the dose of ASM can be used as a measurement of anacute phase response. In certain embodiments, a plasma fibrinogenconcentration of greater than approximately 350 mg/dL, 375 mg/dL, 400mg/dL, 425 mg/dL, or 450 mg/dL, or in the range of 350 mg/dL to 400mg/dL, 350 mg/dL to 450 mg/dL or 400 mg/dL to 450 mg/dL 6 hours, 8hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours or 72hours after administration of the dose of ASM can be used as ameasurement of an acute phase response.

In one embodiment, an increase in the percentage of neutrophils of totalwhite blood cells 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24hours, 36 hours, 48 hours or 72 hours after administration of a dose ofASM relative to a patient's percentage of neutrophils of total whiteblood cells prior to administration of ASM can be utilized as ameasurement of an acute phase response. In another embodiment, anincrease in the percentage of neutrophils of total white blood cellsthat is greater than the normal percentage of neutrophils of total whiteblood cells concentration 6 hours, 8 hours, 12 hours, 16 hours, 18hours, 24 hours, 36 hours, 48 hours or 72 hours after administration ofthe dose of ASM can be utilized as a measurement of an acute phaseresponse. In certain embodiments, an increase in the percentage ofneutrophils of total white blood cells that is 70%, 75%, 80%, 85%, 90%,95% or greater 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours,36 hours, 48 hours or 72 hours after administration of the dose of ASMcan be utilized as a measurement of an acute phase response.

As used herein, the term “low, non-toxic dose(s)” and the like in thecontext of the initial dose or doses administered to a subject refers toa dosage that is the first dose or doses of ASM administered to asubject to treat ASMD that is non-toxic. In certain embodiments, a low,non-toxic dose(s) is a dose of 0.001 mg/kg to 0.01 mg/kg, 0.001 mg/kg to0.01 mg/kg, 0.001 mg/kg to 0.05 mg/kg, 0.001 mg/kg to 0.1 mg/kg, 0.001mg/kg to 0.5 mg/kg, 0.05 mg/kg to 0.275 mg/kg, 0.075 mg/kg to 0.275mg/kg, 0.05 mg/kg to 0.2 mg/kg, 0.075 mg/kg to 0.2 mg/kg, 0.1 mg/kg to0.275 mg/kg, 0.1 mg/kg to 0.25 mg/kg, 0.1 mg/kg to 1 mg/kg, 0.5 mg/kg to1 mg/kg, 0.75 mg/kg to 1 mg/kg, 0.1 mg/kg to 2 mg/kg, 0.5 mg/kg to 2mg/kg, 0.75 mg/kg to 2 mg/kg, 1 mg/kg to 2 mg/kg or 1.25 mg/kg to 2mg/kg, 1.5 mg/kg to 2 mg/kg or 1.75 mg/kg to 2 mg/kg of ASM. In somespecific embodiments, a low, non-toxic dose is a dose of 0.001 mg/kg,0.005 mg/kg, 0.0075 mg/kg, 0.01 mg/kg, 0.0125 mg/kg, 0.025 mg/kg, 0.05mg/kg, 0.075 mg/kg, 0.1 mg/kg, 0.125 mg/kg, 0.15 mg/kg, 0.175 mg/kg, 0.2mg/kg, 0.225 mg/kg, 0.25 mg/kg, 0.275 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.75 mg/kg, 0.8 mg/kg, 0.9 mg/kg or 1 mg/kgof ASM.

The terms “subject” and “patient” are used herein interchangeably torefer to a human. In a specific embodiment, the human has or has beendiagnosed as having an ASMD.

As used herein, the term “therapeutically effective” in the context ofadministering a dose of ASM to a subject refers to the amount of ASMthat results in a beneficial or therapeutic effect. In specificembodiments, the term “therapeutically effective” in context ofadministering a dose of ASM to a subject refers to the amount of ASMrefers which is sufficient to achieve at least one, two, three, four ormore of the following effects: (i) a reduction in spleen volume asassessed by techniques known in the art, e.g., MRI; (ii) a reduction inliver sphingomyelin levels as assessed by techniques known in the art,e.g., biochemical analysis and/or histomorphometric analysis of liversamples; (iii) an increase in exercise capacity as assessed bytechniques known in the art, e.g., maximum workload by cycle erogmetry,including percent predicted maximum workload, peak oxygen consumptionand carbon dioxide production; (iv) an increase in pulmonary function asassessed by techniques known in the art, e.g., techniques described inAmerican Thoracic Society, 1991, Am. Rev. Respir. Dis. 144: 1202-1218,such as diffusing capacity (DLco), percent predicted forced vitalcapacity (FVC) as measured by, e.g., spirometric techniques, forcedexpiratory volume within 1 second (FEV₁) as measured by, e.g.,spirometric techniques, and total lung capacity; (v) a decrease inbronchial alveolar lavage (BAL) sphingomyelin; (vi) a decrease in livervolume as assessed by techniques known in the art, e.g., MM: (vii) animprovement in lung appearance as assessed by techniques known in theart, e.g., high resolution CT scan or chest X-ray; (viii) a decrease insphinomyelin concentration in the skin, plasma and dried blood spot(DBS) as measured by, e.g., tandem mass spectrometry; (ix) a reductionor the amelioration of the severity of ASMD and/or a symptom associatedtherewith; (x) a reduction in the duration of a symptom associated withASMD; (xi) the prevention in the recurrence of a symptom associated withASMD; (xii) a reduction in hospitalization of a subject; (vi) areduction in hospitalization length; (xiii) an increase in the survivalof a subject; (xiv) a reduction in mortality; (xv) a decrease inhospitalization rate; (xvi) a reduction in the number of symptomsassociated with ASMD; (xvii) an increase in symptom-free survival ofASMD patients; (xviii) an improvement in neurological function (e.g.,psychomotor function, social responsiveness, etc.); (xix) an improvementin lung clearance as measured by, e.g., BAL cell count and profile; (xx)a decrease in serum levels of chitotriosidase; (xxi) a decrease in serumlevels of CCL18; (xxii) an improvement in lipid profile (e.g., HDL, LDL,cholesterol, triglycerides, and total cholesterol:HDL ratio); and(xxiii) improved quality of life as assessed by, e.g., a questionnaire.

As used herein, the terms “therapies” and “therapy” can refer to anyprotocol(s), method(s), compositions, formulations, and/or agent(s) thatcan be used in the treatment, management, or amelioration of ASMD orcondition or symptom associated therewith. In certain embodiments, theterms “therapies” and “therapy” refer to biological therapy, supportivetherapy, and/or other therapies useful in treatment, management,prevention, or amelioration of ASMD or condition or symptom associatedtherewith. In embodiments, the term “therapy” refers to a therapy thatdoes one, two or more of the following: (i) enhances the delivery of ASMto sites of pathology, (ii) enhances the activity of ASM, and (iii)enhances the stability of ASM. In certain embodiments, the term“therapy” refers to a therapy other than ASM. In specific embodiments,an “additional therapy” and “additional therapies” refer to a therapyother than ASM.

As used herein, the term “toxic effect(s)” and the like refers one, two,three or all of the following subsequent to the administration of adose(s) of ASM: (i) a moderate or severe related adverse event asdefined by a clinical symptom that interferes with normal dailyfunctioning and requires additional monitoring, intervention, ortreatment, or, an abnormal laboratory value or procedural result ofclinical concern that requires further monitoring, treatment, orinvestigating. See, e.g., the Clinical Data Interchange StandardsConsortium Study Data Tabulation Model standard terminology v.3.1.1;(ii) a total bilirubin value of greater than 1.5 mg/dL, 1.75 mg/dL, 2.0mg/dL, 2.1 mg/dL, 2.2 mg/dL, 2.3 mg/dL, 2.4 mg/dL, 2.5 mg/dL, 2.6 mg/dL,2.7 mg/dL, 2.75 mg/dL, 2.8 mg/dL, 2.9 mg/dL, 3.0 mg/dL, 3.1 mg/dL, 3.2mg/dL, 3.3 mg/dL, 3.4 mg/dL, 3.5 mg/dL, 3.6 mg/dL, 3.7 mg/dL, 3.8 mg/dL,3.9 mg/dL or 4 mg/dL or in the range of 2.1 mg/dL to 2.5 mg/dL, 2.1mg/dL to 3.0 mg/dL, or 2.1 mg/dL to 4 mg/dL that lasts for greater than16 hours, 18 hours, 24 hours, 36 hours, 48 hours or 72 hours, 5 days,one week, two weeks or three weeks after administration of the dose ofASM; (iii) a plasma ceramide concentration of greater than 8.2 μg/mL,8.3 μg/mL, 8.4 μg/mL, 8.5 μg/mL, 8.75 μg/mL, 9 μg/mL, 9.5 μg/mL, 10μg/mL, 11 μg/mL, 12 μg/mL, 13 μg/mL, 14 μg/mL, 15 μg/mL, 16 μg/mL, 17μg/mL, 18 μg/mL, 19 μg/mL, 20 μg/mL, 25 μg/mL, 30 μg/mL, 35 μg/mL, 40μg/mL, 45 μg/mL, 50 μg/mL, 55 μg/mL, 60 μg/mL, 65 μg/mL, 70 μg/mL, 75μg/mL, or 80 μg/mL, or in the range of 8.2 μg/mL to 10 μg/mL, 8.5 μg/mLto 10 μg/mL, 9 μg/mL to 12 μg/mL, 10 μg/mL to 12 μg/mL, 10 μg/mL to 15μg/mL, 10 μg/mL to 20 μg/mL, 15 μg/mL to 20 μg/mL, or 20 μg/mL to 30μg/mL 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 18 hours, 24hours, 36 hours, 48 hours or 72 hours after administration of the doseof ASM; or (iv) an acute phase response.

An acute phase response can be assessed by a change in the concentrationof an acute phase reactant (such as, e.g., C-reactive protein, ferritin,albumin, IL-8, 11-6, calcitonin, fibrinogen, iron or transferrin), achange in the percentage of neutrophils, a change in prothrombin time,or a change in partial thromboplastin time. In a specific embodiment, anincrease in CRP/hs-CRP concentration 6 hours, 8 hours, 12 hours, 18hours, 24 hours, 36 hours, 48 hours or 72 hours after administration ofa dose of ASM relative to a patient's CRP/hs-CRP concentration prior toadministration of ASM can be utilized as a measurement of an acute phaseresponse. In another specific embodiment, a plasma CRP/hs-CRPconcentration that is greater than the normal plasma CRP/hs-CRPconcentration 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours,36 hours, 48 hours or 72 hours after administration of the dose of ASMcan be utilized as a measurement of an acute phase response. In certainembodiments, a plasma CRP/hs-CRP concentration of greater thanapproximately 8.1 mg/L, 8.2 mg/L, 8.3 mg/L, 8.4 mg/L, 8.5 mg/L, 8.6mg/L, 8.7 mg/L, 8.8 mg/L, 8.9 mg/L, 9 mg/L, 9.5 mg/L, 10 mg/L, 11 mg/L,or 12 mg/L, or in the range of 8.5 mg/L to 10 mg/L, or 8.5 mg/L to 12mg/L, or 10 mg/L to 12 mg/L 6 hours, 8 hours, 12 hours, 16 hours, 18hours, 24 hours, 36 hours, 48 hours or 72 hours after administration ofthe dose of ASM can be used as a measurement of an acute phase response.

In a specific embodiment, an increase in ferritin concentration 6 hours,8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours or72 hours after administration of a dose of ASM relative to a patient'sferritin concentration prior to administration of ASM can be used as ameasurement of an acute phase response. In another specific embodiment,a plasma ferritin concentration that is greater than the normal plasmaferritin concentration 6 hours, 8 hours, 12 hours, 18 hours, 24 hours,36 hours, 48 hours or 72 hours after administration of the dose of ASMcan be used as a measurement of an acute phase response. In certainembodiments, a plasma ferritin concentration of greater thanapproximately 600 ng/mL, 625 ng/mL, 650 ng/mL, 675 ng/mL, 700 ng/mL, 725ng/mL, 750 ng/mL, 775 ng/mL, 800 ng/mL, 850 ng/mL, 900 ng/mL, 950 ng/mL,1000 ng/mL, 1050 ng/mL, 1100 ng/mL, 1150 ng/mL, or 1200 ng/mL or in therange of 600 ng/mL to 800 ng/mL, 650 ng/mL to 850 ng/mL, 600 ng/mL to1000 ng/mL, 600 ng/mL to 1200 ng/mL, 800 ng/mL to 1000 ng/mL, 900 ng/mLto 1000 ng/mL, or 1000 ng/mL to 1200 ng/mL 6 hours, 8 hours, 12 hours,16 hours, 18 hours, 24 hours, 36 hours, 48 hours or 72 hours afteradministration of the dose of ASM can be used as a measurement of anacute phase response.

In a specific embodiment, an increase in fibrinogen concentration 6hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48hours or 72 hours after administration of a dose of ASM relative to apatient's fibrinogen concentration prior to administration of ASM can beused as a measurement of an acute phase response. In another specificembodiment, a plasma fibrinogen concentration that is greater than thenormal plasma fibrinogen concentration 6 hours, 8 hours, 12 hours, 16hours, 18 hours, 24 hours, 36 hours, 48 hours or 72 hours afteradministration of the dose of ASM can be used as a measurement of anacute phase response. In certain embodiments, a plasma fibrinogenconcentration of greater than approximately 350 mg/dL, 375 mg/dL, 400mg/dL, 425 mg/dL, or 450 mg/dL, or in the range of 350 mg/dL to 400mg/dL, 350 mg/dL to 450 mg/dL or 400 mg/dL to 450 mg/dL 6 hours, 8hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours or 72hours after administration of the dose of ASM can be used as ameasurement of an acute phase response.

In a specific embodiment, a decrease in albumin concentration 6 hours, 8hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours or 72 hoursafter administration of a dose of ASM relative to a patient's albuminconcentration prior to administration of ASM can be utilized as ameasurement of an acute phase response. In certain embodiments, adecrease in the following magnitude of albumin concentration of 0.2,0.4, 0.6, 1, 1.5, 2.0 g/dL from a normal range of 3.5 to 5.0 g/dL 6hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48hours or 72 hours after administration of the dose of ASM can be used asa measurement of an acute phase response.

In a specific embodiment, a decrease in ferritin concentration 6 hours,8 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours or 72 hoursafter administration of a dose of ASM relative to a patient's ferritinconcentration prior to administration of ASM can be utilized as ameasurement of an acute phase response. In certain embodiments, adecrease in the following magnitude of ferritin concentration of 20, 40,60, 80, 100, 120, 140, 160 mcg/dL from a normal range of 60 to 170mcg/dL 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36hours, 48 hours or 72 hours after administration of the dose of ASM canbe used as a measurement of an acute phase response.

In a specific embodiment, a decrease in transferrin concentration 6hours, 8 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours or 72hours after administration of a dose of ASM relative to a patient'stransferrin concentration prior to administration of ASM can be utilizedas a measurement of an acute phase response. In certain embodiments, adecrease in the following magnitude of transferrin concentration of 20,40, 60, 80, 100, 120, 140, 160, 180 mg/dL from a normal range of 202 to336 mg/dL 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36hours, 48 hours or 72 hours after administration of the dose of ASM canbe used as a measurement of an acute phase response.

In one embodiment, an increase in the percentage of neutrophils of totalwhite blood cells 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24hours, 36 hours, 48 hours or 72 hours after administration of a dose ofASM relative to a patient's percentage of neutrophils of total whiteblood cells prior to administration of ASM can be utilized as ameasurement of an acute phase response. In another embodiment, anincrease in the percentage of neutrophils of total white blood cellsthat is greater than the normal percentage of neutrophils of total whiteblood cells concentration 6 hours, 8 hours, 12 hours, 16 hours, 18hours, 24 hours, 36 hours, 48 hours or 72 hours after administration ofthe dose of ASM can be utilized as a measurement of an acute phaseresponse. In certain embodiments, an increase in the percentage ofneutrophils of total white blood cells that is 70%, 75%, 80%, 85%, 90%,95% or greater 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours,36 hours, 48 hours or 72 hours after administration of the dose of ASMcan be utilized as a measurement of an acute phase response.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphic representation of the protocol design.

FIG. 2 is a chart showing the demography and baseline characteristics ofthe patients enrolled in the protocol described below.

FIGS. 3A, 3B, and 3C are graphs depicting the plasma levels of ceramide(FIG. 3A) and the plasma levels of sphingomyelin (FIG. 3B), and thesphingomyelin level in dried blood spot (FIG. 3C) over time in differentpatients being administered different doses of rhASM. The right axis ofthe graphs depict the patient number and the dosage of rhASM.

FIG. 4 is a graph depicting the total bilirubin levels determined overtime in different patients being administered different doses of rhASMduring the protocol. The right axis of the graph depicts the patientnumber and the dosage of rhASM.

FIGS. 5A-5G are graphs depicting the levels of CRP/hs-CRP (FIG. 5A),percent neutrophils (FIG. 5B), fibrinogen (FIG. 5C), ferritin (FIG. 5D),IL-8 (FIG. 5E), IL-6 (FIG. 5F), and calcitonin (FIG. 5G) determined overtime in different patients being administered different doses of rhASMduring the protocol. The right axis of the graphs depict the patientnumber and the dosage of rhASM.

FIG. 6 is a chart of treatment emergent adverse events for four patientseach on a different dose of rhASM, which events were considered to berelated (possibly, probably, or definitely) to treatment.

FIG. 7 is a diagram showing the primary treatment period, consistingof—dose escalation and dose maintenance phases, of the Phase 2 rhASMrepeat dose protocol in patients with ASMD.

5. DETAILED DESCRIPTION OF THE INVENTION

The invention relates to dose escalation enzyme replacement therapy forthe treatment of human subjects having ASMD—particularly subjects havingnon-neurological manifestations of NPD, and in certain embodiments, NPDtype B. More particularly, the enzyme, ASM, is administered to suchpatients at an initial low, non-toxic dose that is then escalated insubsequent administrations. The highest dose of ASM tolerated by thepatient can then be used as a maintenance dose. Alternatively, atherapeutically effective dose less than the highest dose tolerated canbe used as a maintenance dose.

Treatment of NPD requires doses high enough to achieve adequatedistribution of the ASM enzyme in organs of pathology (e.g., the spleen,lungs, heart, kidney and brain). Following intravenous administration ofrecombinant human ASM in ASMKO mice, most of the ASM activitydistributes to the liver with small amounts of ASM enzymatic activitydetected in other organs of pathology, such as the spleen, heart, kidneyand lung (see, e.g., FIG. 9B of He et al., 1999, Biochimia et BiophsyicaActa 1432: 251-264). Thus very high doses would be required to ensuredistribution and delivery of the administered enzyme to the lung, heartand kidney in patients afflicted with ASMD or Niemann-Pick disease.

Studies in an ASM knockout mouse model (ASKMO mice) showed that themajority of rhASM administered distributes to the liver and spleen whereit reduces substrate, but to a much lesser extent in lungs, heart andbrain (Miranda et al. FASEB 2000, 14:1988). In subsequent studies usinghigher doses of rhASM in the ASMKO mouse model, substrate was reducedand toxicity was not observed at doses ≤3.0 mg/kg; in fact, clinicalsymptoms of toxicity were not observed until doses ≥10 mg/kg were used.See, “Dose Responsive Toxicological Findings Following IntravenousAdministration of Recombinant Human Acid Sphingomyelinase (rhASM) toAcid Sphingomyelinase Knock-out (ASMKO) Mice,” C. Nickerson, J. Murray,A. Vitsky, M. Hawes, S. Ryan, P. Ewing, B. Thurberg, L. Andrews. DeptPharm/Tox, Pathology, Genzyme Corp., Framingham, Mass., American Societyof Human Genetics 2005; and “Elevations of Pro-Inflammatory Cytokinesand Decreases in Cardiovascular Hemodynamics Following IntravenousAdministration of Recombinant Human Acid Sphingomyelinase (rhASM) toAcid Sphingomyelinase Knock-out (ASMKO) Mice,” J. Murray, A. M.D'Angona, C. Nickerson, A. Vitsky, M. Hawes, S. Ryan, P. Ewing, B.Thurberg, L. Andrews. Dept. Pharmacology/Toxicology & Pathology, GenzymeCorp., Framingham, Mass., Society of Toxicology 2006

Based on these ASKMO data, we treated non-neuronopathic ASMD humansubjects with a conservative maximum dose of 1.0 mg/kg as described inSection 6, infra. Quite unexpectedly, toxicity in the human subjects,including the onset of adverse events with clinical symptoms, wasobserved using doses as low as 0.3 mg/kg! This result was especiallysurprising, since the ASM enzyme is absent in the knock out mouse modelwhich should reflect a more severe condition than the human subjects whohave at least some enzyme activity and relatively mild disease.

Without being bound by any theory, the administration of high doses ofASM to NPD patients can result in the hydrolysis of large amounts ofsphingomyelin into large concentrations of ceramide, which may producethe toxic side effects observed in those NPD patients. The ASM enzymehydrolyzes sphingomyelin, which is a major component of the plasmamembrane of cells (see, e.g., Milaus et al., 2010 FEBS Letters 584:1887-1894), into ceramide and phosphocholine. Ceramide is known to playa role in cell death and is known to be a pro-apoptotic agent (see,e.g., Smith and Schuchman, 2008, FASEB 22: 3419-3431).

Moreover, unlike other lysosomal enzymes associated with characterizedlysosomal storage diseases, ASM hydrolyzes sphingomyelin at the neutralpH found in the plasma and the acidic pH found in the lysosome (see,e.g., Schissel et al., 1998. J. Biol. Chem. 273: 2738-2746). The abilityof the ASM enzyme to function in the plasma can result in the hydrolysisof sphingomyelin found in lipoproteins and the plasma membrane of cells,which can increase the amount of the breakdown product, ceramide, whichmay cause the toxic side effects observed in NPD patients administeredhigh doses of ASM enzyme.

To solve the problem of achieving adequate distribution of the ASMenzyme to the organs of pathology while avoiding or minimizing thetoxicity associated with administering high doses of the enzyme, theinventors develop the regimens described herein, in which a low,non-toxic dose of the ASM enzyme is initially administered to a NPDpatient and the dose is escalated over time. As the dose of the ASMenzyme is escalated, the patient can be monitored fortotal/direct/indirect bilirubin concentrations, the production of acutephase reactants, the production of inflammatory mediators, and relatedadverse events. The administration of a low dose of ASM and theescalation of the enzyme facilitates the debulking of the accumulatedsphingomyelin. Once the patient is debulked, higher doses of the ASMenzyme may be safely administered to the patient to ensure adequatedistribution of the ASM enzyme to target organs (e.g., liver, spleen,lungs, heart, kidney, brain, bone marrow, skeleton, joints, etc.). Incertain embodiments, the maximum dose tolerated by the patient can beused as the maintenance dose. Alternatively, a therapeutically effectivedose less than the highest dose tolerated can be used as a maintenancedose. In some embodiments, based upon a patient's condition, themaintenance dose may be increased or decreased.

In certain embodiments, treatment of the patient can be monitored bymeasuring the plasma sphingomyelin levels, plasma ceramide levels, theproduction of “acute phase reactants” and inflammatory mediators thatare a measure of inflammatory responses, bilirubin concentrations(total, direct or indirect) and/or other biochemical markers to ensure astable response before elevating the dose to the next level. Thesemarkers include, but are not limited to CRP/hs-CRP, cytokines (e.g.,IL-8, I1-6), calcitonin, and ferritin. In specific embodiments, thepatient may be monitored for one or more related adverse events whichmay include, but is not limited to constitutional symptoms (e.g., fever,nausea, vomiting, pain, myalgia and jaundice).

5.1. Dose Escalation Protocol

Methods are described for treating ASMD involving the administration ofone or more initial, low non-toxic doses of ASM to a subject to reducethe amount of sphingomyelin that has accumulated in the subject. After acertain period of time, the dose of ASM can be escalated until thehighest dosage tolerated by the subject that is therapeuticallyeffective is achieved. Once this dosage is identified, it can be used asthe maintenance dose to treat the subject. The maintenance dose can beadministered weekly, biweekly, or monthly to the subject to treat ASMD.In some embodiments, a subject receiving a maintenance dose is monitoredevery 3 months, every 6 months or yearly for one, two, three or all ofthe following: (i) a related adverse events; (ii) total/direct/indirectbilirubin concentrations; (iii) plasma ceramide concentration; or (iv)an acute phase response. If the subject experiences a related adverseevent of moderate intensity (e.g., a related moderate adverse event), atotal bilirubin concentration greater than the total bilirubin value fora human without ASMD (e.g., a healthy human), a plasma ceramideconcentration greater than the plasma ceramide concentration of a humanwithout ASMD (e.g., a healthy human), or an acute phase response, thenthe dose administered to the subject can be evaluated by a physician orother medical professional to determine whether the dose should beadjusted.

In one embodiment, a method for treating a human subject having an acidsphingomyelinase deficiency, comprises: (a) a regimen for debulkingaccumulated sphingomyelin substrate in the human subject comprising: (i)administering an initial low non-toxic dose of ASM to the human subject;(ii) administering successively higher doses of ASM to the humansubject, and monitoring the subject for one or more adverse side effectsafter each successive dose as indicated by elevated bilirubin or arelated adverse event; and (b) a maintenance regimen comprisingadministering a dose equal to or less than the highest dose tolerated bythe subject as the maintenance dose for the subject. In certainembodiments, the initial dose ranges is from 0.1 mg/kg to 0.5 mg/kg or0.1 mg/kg to 1 mg/kg of ASM. In some embodiments, the successivelyhigher doses are administered one, two, three or four weeks after theprevious dose. In certain embodiments, the successively higher dose isapproximately 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 1.2 mg/kg, 1.5 mg/kg,1.75 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg or 5 mg/kg higher than theprevious dose. In some embodiments, the successively higher dose is 0.1to 0.5 mg/kg, 0.1 mg/kg to 1 mg/kg, 0.5 mg/kg to 1 mg/kg, 0.5 mg/kg to 2mg/kg, 1 mg/kg to 2 mg/kg, 2 mg/kg to 4 mg/kg or 2 mg/kg to 5 mg/kghigher than the previous dose. In certain embodiments, the highest dosetolerated by the subject is 1 mg/kg to 2.5 mg/kg. In some embodiments,the highest dose is administered to the human subject as a maintenancedose.

In certain embodiments, a method for treating a human subject havingASMD, comprises: (a) a debulking ASM administration to reduce the amountof sphingomyelin that has accumulated in the human subject, wherein thedebulking ASM administration comprises: (i) administering a low,non-toxic dose of ASM to the human subject; and (ii) administeringsuccessively higher doses of ASM to the human if the human subject doesnot manifest one or more adverse side effects as indicated by elevatedtotal bilirubin concentration, elevated plasma ceramide concentration,the production of acute phase reactants, the production of inflammatorymediators, or an adverse event (e.g., such as defined by the ClinicalData Interchange Standards Consortium Study Data Tabulation Modelstandard terminology v.3.1.1); and (b) a maintenance ASM administration,wherein the maintenance ASM administration comprises repeatedadministration of a maintenance dose of ASM to the human subject. Insome embodiments, the patient is monitored for a period of time afteradministration of a dose of ASM (e.g., 6 hours, 12 hours, 16 hours, 24hours, 48 hours, 72 hours, weekly, or up until the next dose) for one ormore adverse side effects or total bilirubin. In certain embodiments,the maintenance dose that is administered may be adjusted during thecourse of treatment of the patient. In some embodiments, the maintenancedose administered to the subject is the highest dose tolerated by thesubject.

In a particular embodiment, a method for treating ASMD comprisesadministering to a subject in need thereof an initial low, non-toxicdose of ASM (e.g., a dose of 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg,0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 0.1mg/kg to 0.5 mg/kg, or 0.5 to 1 mg/kg of ASM) and after a certain periodof time (e.g., 3 days, 1 week, 2 weeks, or 3 weeks) successivelyincreasing the dose of ASM administered to the subject until the levelof ASM activity in one or more organs of pathology is at least 5%, 6%,7%, 8%, 9%, 10%, 12%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 80%, 85%,90%, 95% or more of the activity of ASM in the corresponding organ in asubject(s) without ASMD (e.g., a healthy subject or population of 5, 10,15, 20, 30, 40, 50, 75, 100, 150, 175 or more subjects) as measured bytechniques known in the art, such as, e.g., the technique described inHe et al., 2003, Analytical Biochemistry 314: 116-120. In anotherembodiment, a method for treating ASMD comprises administering to asubject in need thereof a dose of 0.1 mg/kg of ASM and after a certainperiod of time (e.g., 3 days, 1 week, 2 weeks, or 3 weeks) successivelyincreasing the dose of ASM administered to the subject until the levelof ASM activity in one or more of the following organs of pathology is5% to 10%, 5% to 15%, 5% to 20%, 10% to 15%, 10% to 20%, 15% to 20%, 15%to 25%, 25% to 50%, 50% to 75%, or 75% to 95% of the normal activity ofASM in the corresponding organ in a subject(s) without ASMD (e.g., ahealthy subject or population of 5, 10, 15, 20, 30, 40, 50, 75, 100,150, 175 or more subjects) as measured by techniques known in the art,such as, e.g., the technique described in He et al., 2003, AnalyticalBiochemistry 314: 116-120. In certain embodiments, the dose issuccessively increased if the total bilirubin concentration is less thanor equal to 2.0 mg/dL or 2.1 mg/dL and the subject does not experience amoderate or severe related adverse event. In specific embodiments, theactivity of ASM in normal healthy subjects is estimated to beapproximately 20 to 40 units/mg of protein for the brain, heart, kidney,and liver based upon the activity of ASM in similar organs in healthymice using the assay described in Horinouchi et al., 1995, NatureGenetics 10: 288-293 (which is incorporated by reference herein in itsentirety). In certain embodiments, the activity of ASM in normal healthysubjects is estimated to be approximately 15 to 25 units/mg of proteinfor the lung and 10 to 15 units/mg of protein for the spleen based uponthe activity of ASM in similar organs in healthy mice using the assaydescribed in Horinouchi et al., 1995, Nature Genetics 10: 288-293. Incertain embodiments, once the ASM activity reaches normal or a certainpercentage of normal in one or more organs of pathology, a dose equal toor less than the highest dose tolerated by the subject can beadministered to the subject as the maintenance dose. Over time, themaintenance dose may be adjusted depending the health of the subject.Depending upon a subject's circumstances, the maintenance dose may beincreased or decreased.

In one embodiment, a method for treating ASMD comprises: (a)administering to a human in need thereof an initial low, non-toxic doseof ASM; and (b) administering successively higher doses of ASM if thehuman does not manifest one, two, three or four of the followingside-effects after the administration of a dose of ASM: (i) a severerelated adverse event as defined by, e.g., the Clinical Data InterchangeStandards Consortium Study Data Tabulation Model standard terminologyv.3.1.1; (ii) a total bilirubin value of greater than 1.5 mg/dL, 1.75mg/dL, 2.0 mg/dL, 2.1 mg/dL, 2.2 mg/dL, 2.3 mg/dL, 2.4 mg/dL, 2.5 mg/dL,2.6 mg/dL, 2.7 mg/dL, 2.75 mg/dL, 2.8 mg/dL, 2.9 mg/dL, 3.0 mg/dL, 3.1mg/dL, 3.2 mg/dL, 3.3 mg/dL, 3.4 mg/dL, 3.5 mg/dL, 3.6 mg/dL, 3.7 mg/dL,3.8 mg/dL, 3.9 mg/dL or 4 mg/dL, or in the range of 2.1 mg/dL to 2.5mg/dL, 2.1 mg/dL to 3.0 mg/dL, or 2.1 mg/dL to 4 mg/dL that lasts forgreater than 18 hours, 24 hours, 36 hours, 48 hours or 72 hours, 5 days,one week, two weeks or three weeks after administration of the dose ofASM; (iii) a plasma ceramide concentration of greater than 8.2 μg/mL,8.3 μg/mL, 8.4 μg/mL, 8.5 μg/mL, 8.75 μg/mL, 9 μg/mL, 9.5 μg/mL, 10μg/mL, 11 μg/mL, 12 μg/mL, 13 μg/mL, 14 μg/mL, 15 μg/mL, 16 μg/mL, 17μg/mL, 18 μg/mL, 19 μg/mL, 20 μg/mL, 25 μg/mL, 30 μg/mL, 35 μg/mL, 40μg/mL, 45 μg/mL, 50 μg/mL, 55 μg/mL, 60 μg/mL, 65 μg/mL, 70 μg/mL, 75μg/mL, or 80 μg/mL, or in the range of 8.2 μg/mL to 10 μg/mL, 8.5 μg/mLto 10 μg/mL, 9 μg/mL to 12 μg/mL, 10 μg/mL to 12 μg/mL, 10 μg/mL to 15μg/mL, 10 μg/mL to 20 μg/mL, 15 μg/mL to 20 μg/mL, or 20 μg/mL to 30μg/mL 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 18 hours, 24hours, 36 hours, 48 hours or 72 hours after administration of the doseof ASM; or (iv) an acute phase response. In accordance with thisembodiment, the human in need thereof can be administered successivelyhigher doses of ASM so long the human does not manifest any one or moreof items (i) to (iv). In the event that the human manifests any one ormore of items (i) to (iv), then depending on the severity themanifestation, the same dose that resulted in the manifestation of items(i) to (iv) may be repeated or the dose may be decreased to the previousdose.

In another embodiment, a method for treating ASMD comprises: (a)administering to a human in need thereof an initial dose of 0.1 mg/kg ofASM; (b) monitoring one or more of the following in the human subsequentthe administration of the dose of ASM: (i) total bilirubinconcentration, (ii) the manifestation of a related adverse event; (iii)an acute phase response; or (iv) plasma ceramide concentration; (c)determining whether to adjust (e.g., increase or decrease) or maintainthe dose of ASM based on one or more of items (i) to (iv); and (d)repeating steps (b) and (c) following the administration of the dose ofASM determined in the previous step (c). In another embodiment, a methodfor treating ASMD comprises: (a) administering to a human in needthereof two initial doses of 0.1 mg/kg of ASM 2 to 4 weeks apart; (b)monitoring one or more of the following in the human subsequent to theadministration of each dose of ASM: (i) total bilirubin concentration,(ii) the manifestation of a related adverse event; (iii) an acute phaseresponse; or (iv) plasma ceramide concentration; (c) determining whetherto adjust (e.g., increase or decrease) or maintain the dose of ASM basedon one or more of items (i) to (iv); and (d) repeating steps (b) and (c)following the administration of the dose of ASM determined in theprevious step (c). In accordance with these embodiments, the human canbe administered a higher dose of ASM one or more times 2 to 4 weeksapart if the initial dose of 0.1 mg/kg or the adjusted dose determinedin step (c) results in: (i) a total bilirubin concentration of less thanor equal to 2.0 mg/dL before the deadline for administering another doseof ASM; (ii) no related adverse events or only mild related adverseevents; (iii) a plasma ceramide within the normal range 6 hours, 8hours, 12 hours, 16 hours, 18 hours, 24 hours, 48 hours, or 72 hoursadministration the last dose of ASM; or (iv) no acute phase response oran acute phase response that is not statistically significant. However,the dose can be maintained or decreased if the initial dose of 0.1 mg/kgor the adjusted dose determined in step (c) results in: (i) a totalbilirubin concentration of 2.1 mg/dL or greater before the deadline foradministering another dose of ASM; (ii) a related adverse event; (iii) aplasma ceramide above the normal range 6 hours, 8 hours, 12 hours, 16hours, 18 hours, 24 hours, 48 hours, or 72 hours administration the lastdose of ASM; or (iv) an acute phase response that is statisticallysignificant.

In another embodiment, a method for treating ASMD comprises: (A)administering to a human in need thereof two initial doses of 0.1 mg/kg2 weeks apart; (B) monitoring either (i) total bilirubin concentration,(ii) the manifestation of a related adverse event, (iii) both (i) and(ii) in the human subsequent to the administration of each initial doseof ASM; (C) determining whether to adjust (e.g., increase or decrease)or maintain the dose of ASM based on one or more of items (i) to (iii),and (D) repeating steps (B) and (C) following the administration of thedose of ASM determined in the previous step (C), wherein (a) the doseadministered to the human is increased if the total bilirubinconcentration is less than or equal to 2.0 mg/dL or the human presentswith a mild related adverse event; (b) the human continues to receivethe current dose 1 to 4 times 2 to 4 weeks apart if the total bilirubinconcentration is 2.1 mg/dL to 3.1 mg/dL or the human presents with amoderate related adverse event and this dose is maintained if the totalbilirubin concentration remains greater than 2.0 mg/dL subsequent to thelast administered dose; (c) the dose administered to the human isdecreased or no longer administered ASM if the total bilirubinconcentration is greater than 3 mg/dL or the human presents with severerelated adverse events.

In another embodiment, a method for treating ASMD comprises: (A)administering to a human in need thereof two initial doses of 0.3 mg/kg2 weeks apart; (B) monitoring either (i) total bilirubin concentration,(ii) the manifestation of a related adverse event, (iii) both (i) and(ii) in the human subsequent to the administration of each dose of ASM;(C) determining whether to adjust (e.g., increase or decrease) ormaintain the dose of ASM based on one or more of items (i) to (iii), and(D) repeating steps (B) and (C) following the administration of the doseof ASM determined in the previous step (C), wherein (a) the doseadministered to the human is increased if the total bilirubinconcentration is less than or equal to 2.0 mg/dL or the human presentswith a mild related adverse event; (b) the human continues to receivethe current dose 1 to 4 times 2 to 4 weeks apart if the total bilirubinconcentration is 2.1 mg/dL to 3.1 mg/dL or the human presents with amoderate related adverse event and this dose is maintained if the totalbilirubin concentration remains greater than 2.0 mg/dL subsequent to thelast administered dose; (c) the dose administered to the human isdecreased or no longer administered ASM if the total bilirubinconcentration is greater than 3 mg/dL or the human presents with severerelated adverse events.

In a specific embodiment, a method for treating ASMD comprisesadministering to a subject in need thereof a dose of 0.1 mg/kg of ASMand after two weeks a dose of 0.3 mg/kg of ASM every two weeks. Inanother specific embodiment, a method for treating ASMD comprisesadministering to a subject in need thereof a dose of 0.1 mg/kg of ASM, adose of 0.3 mg/kg of ASM two weeks after the administration of the 0.1mg/kg dose of ASM, and a dose of 0.6 mg/kg of ASM two weeks after theadministration of the dose of 0.3 mg/kg of ASM. In another specificembodiment, a method for treating ASMD comprises administering to asubject in need thereof a dose of 0.1 mg/kg of ASM, a dose of 0.3 mg/kgof ASM two weeks after the administration of the 0.1 mg/kg dose of ASM,a dose of 0.6 mg/kg of ASM two weeks after the administration of thedose of 0.3 mg/kg of ASM, and a dose of 1 mg/kg of ASM two weeks afterthe administration of the dose of 0.6 mg/kg of ASM. In certainembodiments, each dose may be repeated at least two, and preferably twoto four times before escalating the dose to the next level. Inaccordance with these embodiments, the dose is only escalated if thetotal bilirubin value is equal to or less than 2.0 mg/dL and/or thesubject experiences a mild related adverse event. The dose is notescalated if the total bilirubin value is between 2.1 and 3 mg/dL and/orthe subject experiences a moderate related adverse event. The dose isreduced to a previously tolerated dose if the total bilirubin value isgreater than 3.0 mg/dL of total bilirubin and/or the subject experiencesa serious related adverse event.

In specific embodiments, a patient is treated for ASMD in accordancewith the protocol described in Section 8 or 9 et seq., infra, or ananalogous protocol.

In a specific embodiment, a method for treating ASMD comprisesadministering to a subject in need thereof a dose of 0.1 mg/kg, 0.2mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg., 0.6 mg/kg, 0.7 mg/kg, 0.8mg/kg, 0.9 mg/kg, 1 mg/kg, or 0.1 mg/kg to 0.5 mg/kg, or 0.1 mg/kg to 1mg/kg of ASM and after a certain period of time (e.g., 3 days, 1 week, 2weeks, 3 weeks or 4 weeks) successively increasing the dose of ASMadministered to the subject if total bilirubin concentration is lessthan or equal to 2.1 mg/dL and the subject does not experience amoderate or severe related adverse event. In some embodiments, the doseof ASM is successively increased until the maximum or highest dosetolerated by the subject which is therapeutically effective is achieved.In certain embodiments, such highest or maximum dose tolerated isadministered until such time as the accumulated sphingomyelin in theorgans of pathology is debulked after which a maintenance dose that islower than the maximum dose tolerated which is still therapeuticallyeffective is administered to the subject. In some embodiments, themaintenance dose is scaled back over time as the patient's conditionimproves.

In a specific embodiment, the highest dose tolerated that istherapeutically effective is the highest dosage that is effective in thetreatment of ASMD without causing one, two or more severe relatedadverse events or related adverse events as defined by, e.g., theClinical Data Interchange Standards Consortium Study Data TabulationModel standard terminology v.3.1.1. In another embodiment, the highestdose tolerated that is therapeutically effective is the highest dosagethat is effective in the treatment of ASMD without causing an increasein the total bilirubin concentration relative to a patient's totalbilirubin concentration prior to administration of ASM, which increaselasts for greater than two days, three days, five days, one week, twoweeks, or three weeks. In another embodiment, the highest dose toleratedthat is therapeutically effective is the highest dosage that iseffective in the treatment of ASMD without causing a total bilirubinconcentration of greater than normal total bilirubin concentrations thatlasts for greater than 18 hours, 24 hours, 36 hours, 48 hours or 72hours, 5 days, one week, two weeks or three weeks after administrationof the dose of ASM. The normal total bilirubin concentration in a humanwithout ASMD (e.g., a healthy human) is less than approximately 1.2mg/dL. In certain embodiments, the highest dose tolerated that istherapeutically effective is the highest dosage that is effective in thetreatment of ASMD without causing a total bilirubin concentration thatis greater than approximately 1.5 mg/dL, 1.75 mg/dL, 2.0 mg/dL, 2.1mg/dL, 2.2 mg/dL, 2.3 mg/dL, 2.4 mg/dL, 2.5 mg/dL, 2.6 mg/dL, 2.7 mg/dL,2.75 mg/dL, 2.8 mg/dL, 2.9 mg/dL, 3.0 mg/dL, 3.1 mg/dL, 3.2 mg/dL, 3.3mg/dL, 3.4 mg/dL, 3.5 mg/dL, 3.6 mg/dL, 3.7 mg/dL, 3.8 mg/dL, 3.9 mg/dLor 4 mg/dL or in the range of 2.1 mg/dL to 2.5 mg/dL, 2.1 mg/dL to 3.0mg/dL, or 2.1 mg/dL to 4 mg/dL that lasts for greater than 18 hours, 24hours, 36 hours, 48 hours or 72 hours, 5 days, one week, two weeks orthree weeks after administration of the dose of ASM.

In another embodiment, the highest dose tolerated is the highest dosagethat is effective in the treatment of ASMD without resulting in a plasmaceramide concentration that is greater than the normal plasma ceramideconcentration 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours,36 hours, 48 hours or 72 hours after administration of the dose of ASM.The normal plasma ceramide concentration in a human without ASMD (e.g.,a healthy human) is approximately 1.5 to 8 μg/mL. In certainembodiments, the highest dose tolerated is the highest dosage that iseffective in the treatment of ASMD without resulting in a plasmaceramide concentration of greater than approximately 8.2 m/mL, 8.3μg/mL, 8.4 m/mL, 8.5 m/mL, 8.75 m/mL, 9 μg/mL, 9.5 m/mL, 10 μg/mL, 11μg/mL, 12 μg/mL, 13 μg/mL, 14 μg/mL, 15 μg/mL, 16 μg/mL, 17 μg/mL, 18μg/mL, 19 μg/mL, 20 μg/mL, 25 μg/mL, 30 μg/mL, 35 μg/mL, 40 μg/mL, 45μg/mL, 50 μg/mL, 55 μg/mL, 60 μg/mL, 65 μg/mL, 70 μg/mL, 75 μg/mL, or 80μg/mL, or in the range of 8.2 μg/mL to 10 μg/mL, 8.5 μg/mL to 10 μg/mL,9 μg/mL to 12 μg/mL, 10 μg/mL to 12 μg/mL, 10 μg/mL to 15 μg/mL, 10μg/mL to 20 μg/mL, 15 μg/mL to 20 μg/mL, or 20 μg/mL to 30 μg/mL 6hours, 8 hours, 10 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36hours, 48 hours or 72 hours after administration of the dose of ASM.

In another embodiment, the highest dose tolerated that istherapeutically effective is the highest dosage that is effective in thetreatment of ASMD without causing an acute phase response. An acutephase response can be assessed by a change in the concentration of anacute phase reactant, a change in prothrombin time, a change in partialthromboplastin time, or a change in the percentage of neutrophils. Forexample, an acute phase response can be assessed by an increase in oneor more of the following factors following ASM administration to subjectrelative to those factors prior to administration of ASM to the subjector relative to those factors in a human without ASMD (e.g., a healthyhuman): the percentage of neutrophils, the prothrombin time (PT), thepartial thromboplastin time (PTT), the total bilirubin concentration,C-reactive protein (CRP/hs-CRP) concentration, serum amyloid A (SAA),serum amyloid P component, angiotensin converting enzyme (ACE), ferritinconcentration, IL-6 concentration, IL-8 concentration, calcitoninconcentration, albumin concentration, or fibrinogen concentration. Anacute phase response can also be assessed by a decrease in ironconcentration or albumin concentration following ASM administration to asubject relative to the iron concentration or albumin concentration inthe subject prior to ASM administration or relative to the ironconcentration or albumin concentration in a human without ASMD (e.g., ahealthy human).

In a specific embodiment, the highest dose tolerated that istherapeutically effective is the highest dosage that is effective in thetreatment of ASMD without causing an increase in CRP/hs-CRPconcentration 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours,36 hours, 48 hours or 72 hours after administration of the dose of ASMrelative to a patient's CRP/hs-CRP concentration prior to administrationof ASM. In another specific embodiment, the highest dose tolerated thatis therapeutically effective is the highest dosage that is effective inthe treatment of ASMD without resulting in a plasma CRP/hs-CRPconcentration that is greater than the normal plasma CRP/hs-CRPconcentration 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours,36 hours, 48 hours or 72 hours after administration of the dose of ASM.The normal plasma CRP/hs-CRP concentration in a human without ASMD(e.g., a healthy human) is less than approximately 8 mg/L. In certainembodiments, the highest dose tolerated that is therapeuticallyeffective is the highest dosage that is effective in the treatment ofASMD without causing a plasma CRP/hs-CRP concentration of greater thanapproximately 8.1 mg/L, 8.2 mg/L, 8.3 mg/L, 8.4 mg/L, 8.5 mg/L, 8.6mg/L, 8.7 mg/L, 8.8 mg/L, 8.8 mg/L, 8.9 mg/L, 9 mg/L, 9.5 mg/L, 10 mg/L,11 mg/L, or 12 mg/L, or in the range of 8.5 mg/L to 10 mg/L, or 8.5 mg/Lto 12 mg/L, or 10 mg/L to 12 mg/L 6 hours, 8 hours, 12 hours, 16 hours,18 hours, 24 hours, 36 hours, 48 hours or 72 hours after administrationof the dose of ASM.

In a specific embodiment, the highest dose tolerated that istherapeutically effective is the highest dosage that is effective in thetreatment of ASMD without causing an increase in ferritin concentration6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48hours or 72 hours after administration of the dose of ASM relative to apatient's ferritin concentration prior to administration of ASM. Inanother specific embodiment, the highest dose tolerated that istherapeutically effective is the highest dosage that is effective in thetreatment of ASMD without resulting in a plasma ferritin concentrationthat is greater than the normal plasma ferritin concentration 6 hours, 8hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours or 72hours after administration of the dose of ASM. The normal plasmaferritin concentration in a human without ASMD (e.g., a healthy human)is 10 to 30 ng/mL. In certain embodiments, the highest dose toleratedthat is therapeutically effective is the highest dosage that iseffective in the treatment of ASMD without causing a plasma ferritinconcentration of greater than approximately 300 ng/mL, 325 ng/mL, 350ng/mL, 375 ng/mL, 400 ng/mL, 425 ng/mL, 450 ng/mL, 475 ng/mL, 500 ng/mL,525 ng/mL, 550 ng/mL, 575 ng/mL, 600 ng/mL, 625 ng/mL, 650 ng/mL, 675ng/mL, 700 ng/mL, 725 ng/mL, 750 ng/mL, 775 ng/mL, 800 ng/mL, 850 ng/mL,900 ng/mL, 950 ng/mL, 1000 ng/mL, 1050 ng/mL, 1100 ng/mL, 1150 ng/mL, or1200 ng/mL or in the range of 600 ng/mL to 800 ng/mL, 650 ng/mL to 850ng/mL, 600 ng/mL to 1000 ng/mL, 600 ng/mL to 1200 ng/mL, 800 ng/mL to1000 ng/mL, 900 ng/mL to 1000 ng/mL, or 1000 ng/mL, or 1000 ng/mL to1200 ng/mL 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36hours, 48 hours or 72 hours after administration of the dose of ASM canbe used as a measurement of an acute phase response.

In a specific embodiment, an increase in plasma or serum IL-8concentration 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours,36 hours, 48 hours or 72 hours after administration of a dose of ASMrelative to a patient's IL-8 concentration prior to administration ofASM can be used as a measurement of an acute phase response. In anotherspecific embodiment, a plasma or serum IL-8 concentration that isgreater than the normal plasma IL-8 concentration 6 hours, 8 hours, 12hours, 18 hours, 24 hours, 36 hours, 48 hours or 72 hours afteradministration of the dose of ASM can be used as a measurement of anacute phase response. In certain embodiments, a plasma IL-8concentration of greater than approximately 24 pg/mL, 50 pg/mL, 75pg/mL, 100 pg/mL, 200 pg/mL, 300 pg/mL, 400 pg/mL, 500 pg/mL, 600 pg/mL,700 pg/mL, 800 pg/mL, or 900 pg/mL 6 hours, 8 hours, 12 hours, 16 hours,18 hours, 24 hours, 36 hours, 48 hours or 72 hours after administrationof the dose of ASM can be used as a measurement of an acute phaseresponse.

In a specific embodiment, an increase in plasma or serum IL-6concentration 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours,36 hours, 48 hours or 72 hours after administration of a dose of ASMrelative to a patient's IL-6 concentration prior to administration ofASM can be used as a measurement of an acute phase response. In anotherspecific embodiment, a plasma or serum IL-6 concentration that isgreater than the normal plasma or serum IL-6 concentration 6 hours, 8hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours or 72 hoursafter administration of the dose of ASM can be used as a measurement ofan acute phase response. In certain embodiments, a plasma IL-6concentration of greater than approximately 4.4 pg/mL, 6 pg/mL, 8 pg/mL,10 pg/mL, 15 pg/mL, 20 pg/mL, 25 pg/mL, or 30 pg/mL 6 hours, 8 hours, 12hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours or 72 hoursafter administration of the dose of ASM can be used as a measurement ofan acute phase response.

In a specific embodiment, an increase in plasma or serum calcitoninconcentration 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours,36 hours, 48 hours or 72 hours after administration of a dose of ASMrelative to a patient's calcitonin concentration prior to administrationof ASM can be used as a measurement of an acute phase response. Inanother specific embodiment, a plasma or serum calcitonin concentrationthat is greater than the normal plasma calcitonin concentration 6 hours,8 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours or 72 hoursafter administration of the dose of ASM can be used as a measurement ofan acute phase response. In certain embodiments, a plasma calcitoninconcentration of greater than approximately 9.4 pg/mL, 20 pg/mL, 30pg/mL, 40 pg/mL, 50 pg/mL, 75 pg/mL, 100 pg/mL, 150 pg/mL, 200 pg/mL, or250 pg/mL, 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36hours, 48 hours or 72 hours after administration of the dose of ASM canbe used as a measurement of an acute phase response.

In a specific embodiment, the highest dose tolerated that istherapeutically effective is the highest dosage that is effective in thetreatment of ASMD without causing an increase in fibrinogenconcentration 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 36 hours,48 hours or 72 hours after administration of the dose of ASM relative toa patient's fibrinogen concentration prior to administration of ASM. Inanother specific embodiment, the highest dose tolerated that istherapeutically effective is the highest dosage that is effective in thetreatment of ASMD without resulting in a plasma fibrinogen concentrationthat is greater than the normal plasma fibrinogen concentration 6 hours,8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours or72 hours after administration of the dose of ASM. The normal plasmafibrinogen concentration in a human without ASMD (e.g., a healthy human)is 150 mg/dL to 300 mg/dL. In certain embodiments, the highest dosetolerated that is therapeutically effective is the highest dosage thatis effective in the treatment of ASMD without causing a plasmafibrinogen concentration of greater than approximately 350 mg/dL, 375mg/dL, 400 mg/dL, 425 mg/dL, or 450 mg/dL, or in the range of 350 mg/dLto 400 mg/dL, 350 mg/dL to 450 mg/dL or 400 mg/dL to 450 mg/dL, 6 hours,8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours or72 hours after administration of the dose of ASM.

In a specific embodiment, the highest dose tolerated that istherapeutically effective is the highest dosage that is effective in thetreatment of ASMD without causing an increase in the percentage ofneutrophils of total white blood cells 6 hours, 8 hours, 12 hours, 16hours, 18 hours, 24 hours, 36 hours, 48 hours or 72 hours afteradministration of the dose of ASM relative to a patient's percentage ofneutrophils of total white blood cells prior to administration of ASM.In another specific embodiment, the highest dose tolerated that istherapeutically effective is the highest dosage that is effective in thetreatment of ASMD without resulting in a percentage of neutrophils oftotal white blood cells that is greater than the normal percentage ofneutrophils of total white blood cells concentration 6 hours, 8 hours,12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours or 72 hoursafter administration of the dose of ASM. The normal percentage ofneutrophils of total white blood cells in a human without ASMD (e.g., ahealthy human) is 45% to 60%. In certain embodiments, the highest dosetolerated that is therapeutically effective is the highest dosage thatis effective in the treatment of ASMD without causing an increase in thepercentage of neutrophils of total white blood cells that is 70%, 75%,80%, 85%, 90%, 95% or greater 6 hours, 8 hours, 12 hours, 18 hours, 24hours, 36 hours, 48 hours or 72 hours after administration of the doseof ASM.

In another embodiment, the highest dose tolerated that istherapeutically effective is the highest dosage that is effective in thetreatment of ASMD without causing an increase in antibodies to theadministered ASM. In another embodiment, the highest dose tolerated thatis therapeutically effective is the highest dosage that is effective inthe treatment of ASMD without causing a hypersensitivity reaction. Inanother embodiment, the highest dose tolerated that is therapeuticallyeffective is the highest dosage that is effective in the treatment ofASMD without causing cytokine release syndrome.

In certain embodiments, the highest dose tolerated by a subject that istherapeutically effective is 1 mg/kg to 2.5 mg/kg, 2 mg/kg to 3 mg/kg, 3mg/kg to 5 mg/kg, 5 mg/kg to 10 mg/kg, 10 mg/kg to 15 mg/kg, 15 mg/kg to20 mg/kg, 15 mg/kg to 25 mg/kg, 20 mg/kg to 30 mg/kg, or 25 mg/kg to 50mg/kg. In some embodiments, the highest dose tolerated by a subject thatis therapeutically effective is 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg,13 mg/kg, 14 mg/kg, or 15 mg/kg. In certain embodiments, the highestdose tolerated by a subject that is therapeutically effective is 20mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, or 50 mg/kg.

In a specific embodiment, the highest dose tolerated that istherapeutically effective is the highest dosage that is effective in thetreatment of ASMD without causing one, two or more severe relatedadverse events or related adverse events as defined by, e.g., theClinical Data Interchange Standards Consortium Study Data TabulationModel standard terminology v.3.1.1. In another embodiment, the highestdose tolerated that is therapeutically effective is the highest dosagethat is effective in the treatment of ASMD without causing an increasein the total bilirubin concentration relative to a patient's totalbilirubin concentration prior to administration of ASM, which increaselasts for greater than two days, three days, five days, one week, twoweeks, or three weeks. In another embodiment, the highest dose toleratedthat is therapeutically effective is the highest dosage that iseffective in the treatment of ASMD without causing a total bilirubinconcentration of greater than normal total bilirubin concentrations thatlasts for greater than 18 hours, 24 hours, 36 hours, 48 hours or 72hours, 5 days, one week, two weeks or three weeks after administrationof the dose of ASM. The normal total bilirubin concentration in a humanwithout ASMD (e.g., a healthy human) is less than approximately 1.2mg/dL. In certain embodiments, the highest dose tolerated that istherapeutically effective is the highest dosage that is effective in thetreatment of ASMD without causing a total bilirubin concentration thatis greater than approximately 1.5 mg/dL, 1.75 mg/dL, 2.0 mg/dL, 2.1mg/dL, 2.2 mg/dL, 2.3 mg/dL, 2.4 mg/dL, 2.5 mg/dL, 2.6 mg/dL, 2.7 mg/dL,2.75 mg/dL, 2.8 mg/dL, 2.9 mg/dL, 3.0 mg/dL, 3.1 mg/dL, 3.2 mg/dL, 3.3mg/dL, 3.4 mg/dL, 3.5 mg/dL, 3.6 mg/dL, 3.7 mg/dL, 3.8 mg/dL, 3.9 mg/dLor 4 mg/dL or in the range of 2.1 mg/dL to 2.5 mg/dL, 2.1 mg/dL to 3.0mg/dL, or 2.1 mg/dL to 4 mg/dL that lasts for greater than 18 hours, 24hours, 36 hours, 48 hours or 72 hours, 5 days, one week, two weeks orthree weeks after administration of the dose of ASM.

In another embodiment, the highest dose tolerated is the highest dosagethat is effective in the treatment of ASMD without resulting in a plasmaceramide concentration that is greater than the normal plasma ceramideconcentration 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours,36 hours, 48 hours or 72 hours after administration of the dose of ASM.The normal plasma ceramide concentration in a human without ASMD (e.g.,a healthy human) is approximately 1.5 to 8 μg/mL. In certainembodiments, the highest dose tolerated is the highest dosage that iseffective in the treatment of ASMD without resulting in a plasmaceramide concentration of greater than approximately 8.2 m/mL, 8.3μg/mL, 8.4 m/mL, 8.5 m/mL, 8.75 m/mL, 9 μg/mL, 9.5 m/mL, 10 μg/mL, 11μg/mL, 12 μg/mL, 13 μg/mL, 14 μg/mL, 15 μg/mL, 16 μg/mL, 17 μg/mL, 18μg/mL, 19 μg/mL, 20 μg/mL, 25 μg/mL, 30 μg/mL, 35 μg/mL, 40 μg/mL, 45μg/mL, 50 μg/mL, 55 μg/mL, 60 μg/mL, 65 μg/mL, 70 μg/mL, 75 μg/mL, or 80μg/mL, or in the range of 8.2 μg/mL to 10 μg/mL, 8.5 μg/mL to 10 μg/mL,9 μg/mL to 12 μg/mL, 10 μg/mL to 12 μg/mL, 10 μg/mL to 15 μg/mL, 10μg/mL to 20 μg/mL, 15 μg/mL to 20 μg/mL, or 20 μg/mL to 30 μg/mL 6hours, 8 hours, 10 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36hours, 48 hours or 72 hours after administration of the dose of ASM.

In another embodiment, the highest dose tolerated that istherapeutically effective is the highest dosage that is effective in thetreatment of ASMD without causing an acute phase response. An acutephase response can be assessed by a change in the concentration of anacute phase reactant, a change in prothrombin time, a change in partialthromboplastin time, or a change in the percentage of neutrophils. Forexample, an acute phase response can be assessed by an increase in oneor more of the following factors following ASM administration to subjectrelative to those factors prior to administration of ASM to the subjector relative to those factors in a human without ASMD (e.g., a healthyhuman): the percentage of neutrophils, the prothrombin time (PT), thepartial thromboplastin time (PTT), the total bilirubin concentration,C-reactive protein (CRP/hs-CRP) concentration, serum amyloid A (SAA),serum amyloid P component, angiotensin converting enzyme (ACE), ferritinconcentration, albumin concentration or fibrinogen concentration. Anacute phase response can also be assessed by a decrease in ironconcentration or albumin concentration following ASM administration to asubject relative to the iron concentration or albumin concentration inthe subject prior to ASM administration or relative to the ironconcentration or albumin concentration in a human without ASMD (e.g., ahealthy human).

In a specific embodiment, the highest dose tolerated that istherapeutically effective is the highest dosage that is effective in thetreatment of ASMD without causing an increase in CRP/hs-CRPconcentration 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours,36 hours, 48 hours or 72 hours after administration of the dose of ASMrelative to a patient's CRP/hs-CRP concentration prior to administrationof ASM. In another specific embodiment, the highest dose tolerated thatis therapeutically effective is the highest dosage that is effective inthe treatment of ASMD without resulting in a plasma CRP/hs-CRPconcentration that is greater than the normal plasma CRP/hs-CRPconcentration 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours,36 hours, 48 hours or 72 hours after administration of the dose of ASM.The normal plasma CRP/hs-CRP concentration in a human without ASMD(e.g., a healthy human) is less than approximately 8 mg/L. In certainembodiments, the highest dose tolerated that is therapeuticallyeffective is the highest dosage that is effective in the treatment ofASMD without causing a plasma CRP/hs-CRP concentration of greater thanapproximately 8.1 mg/L, 8.2 mg/L, 8.3 mg/L, 8.4 mg/L, 8.5 mg/L, 8.6mg/L, 8.7 mg/L, 8.8 mg/L, 8.9 mg/L, 9 mg/L, 9.5 mg/L, 10 mg/L, 11 mg/L,or 12 mg/L, or in the range of 8.5 mg/L to 10 mg/L, or 8.5 mg/L to 12mg/L, or 10 mg/L to 12 mg/L 6 hours, 8 hours, 12 hours, 16 hours, 18hours, 24 hours, 36 hours, 48 hours or 72 hours after administration ofthe dose of ASM.

In a specific embodiment, the highest dose tolerated that istherapeutically effective is the highest dosage that is effective in thetreatment of ASMD without causing an increase in ferritin concentration6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48hours or 72 hours after administration of the dose of ASM relative to apatient's ferritin concentration prior to administration of ASM. Inanother specific embodiment, the highest dose tolerated that istherapeutically effective is the highest dosage that is effective in thetreatment of ASMD without resulting in a plasma ferritin concentrationthat is greater than the normal plasma ferritin concentration 6 hours, 8hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours or 72hours after administration of the dose of ASM. The normal plasmaferritin concentration in a human without ASMD (e.g., a healthy human)is 10 to 30 ng/mL. In certain embodiments, the highest dose toleratedthat is therapeutically effective is the highest dosage that iseffective in the treatment of ASMD without causing a plasma ferritinconcentration of greater than approximately 600 ng/mL, 625 ng/mL, 650ng/mL, 675 ng/mL, 700 ng/mL, 725 ng/mL, 750 ng/mL, 775 ng/mL, 800 ng/mL,850 ng/mL, 900 ng/mL, 950 ng/mL, 1000 ng/mL, 1050 ng/mL, 1100 ng/mL,1150 ng/mL, or 1200 ng/mL or in the range of 600 ng/mL to 800 ng/mL, 650ng/mL to 850 ng/mL, 600 ng/mL to 1000 ng/mL, 600 ng/mL to 1200 ng/mL,800 ng/mL to 1000 ng/mL, 900 ng/mL to 1000 ng/mL, or 1000 ng/mL to 1200ng/mL 6 hours, 8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36hours, 48 hours or 72 hours after administration of the dose of ASM.

In a specific embodiment, the highest dose tolerated that istherapeutically effective is the highest dosage that is effective in thetreatment of ASMD without causing an increase in fibrinogenconcentration 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 36 hours,48 hours or 72 hours after administration of the dose of ASM relative toa patient's fibrinogen concentration prior to administration of ASM. Inanother specific embodiment, the highest dose tolerated that istherapeutically effective is the highest dosage that is effective in thetreatment of ASMD without resulting in a plasma fibrinogen concentrationthat is greater than the normal plasma fibrinogen concentration 6 hours,8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours or72 hours after administration of the dose of ASM. The normal plasmafibrinogen concentration in a human without ASMD (e.g., a healthy human)is 150 mg/dL to 300 mg/dL. In certain embodiments, the highest dosetolerated that is therapeutically effective is the highest dosage thatis effective in the treatment of ASMD without causing a plasmafibrinogen concentration of greater than approximately 350 mg/dL, 375mg/dL, 400 mg/dL, 425 mg/dL, or 450 mg/dL, or in the range of 350 mg/dLto 400 mg/dL, 350 mg/dL to 450 mg/dL or 400 mg/dL to 450 mg/dL, 6 hours,8 hours, 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours or72 hours after administration of the dose of ASM.

In a specific embodiment, the highest dose tolerated that istherapeutically effective is the highest dosage that is effective in thetreatment of ASMD without causing an increase in the percentage ofneutrophils of total white blood cells 6 hours, 8 hours, 12 hours, 16hours, 18 hours, 24 hours, 36 hours, 48 hours or 72 hours afteradministration of the dose of ASM relative to a patient's percentage ofneutrophils of total white blood cells prior to administration of ASM.In another specific embodiment, the highest dose tolerated that istherapeutically effective is the highest dosage that is effective in thetreatment of ASMD without resulting in a percentage of neutrophils oftotal white blood cells that is greater than the normal percentage ofneutrophils of total white blood cells concentration 6 hours, 8 hours,12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48 hours or 72 hoursafter administration of the dose of ASM. The normal percentage ofneutrophils of total white blood cells in a human without ASMD (e.g., ahealthy human) is 45% to 60%. In certain embodiments, the highest dosetolerated that is therapeutically effective is the highest dosage thatis effective in the treatment of ASMD without causing an increase in thepercentage of neutrophils of total white blood cells that is 70%, 75%,80%, 85%, 90%, 95% or greater 6 hours, 8 hours, 12 hours, 18 hours, 24hours, 36 hours, 48 hours or 72 hours after administration of the doseof ASM.

In another embodiment, the highest dose tolerated that istherapeutically effective is the highest dosage that is effective in thetreatment of ASMD without causing an increase in antibodies to theadministered ASM. In another embodiment, the highest dose tolerated thatis therapeutically effective is the highest dosage that is effective inthe treatment of ASMD without causing a hypersensitivity reaction. Inanother embodiment, the highest dose tolerated that is therapeuticallyeffective is the highest dosage that is effective in the treatment ofASMD without causing cytokine release syndrome.

In certain embodiments, the highest dose tolerated by a subject that istherapeutically effective is 1 mg/kg to 2.5 mg/kg, 2 mg/kg to 3 mg/kg, 3mg/kg to 5 mg/kg, 5 mg/kg to 10 mg/kg, 10 mg/kg to 15 mg/kg, 15 mg/kg to20 mg/kg, 15 mg/kg to 25 mg/kg, 20 mg/kg to 30 mg/kg, or 25 mg/kg to 50mg/kg. In some embodiments, the highest dose tolerated by a subject thatis therapeutically effective is 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg,13 mg/kg, 14 mg/kg, or 15 mg/kg. In certain embodiments, the highestdose tolerated by a subject that is therapeutically effective is 20mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, or 50 mg/kg.

In certain embodiments, a dose of ASM described herein is administeredevery 3 days, every 4 days, every 5 days, every 6 days, every week,every 8 days, every 9 day, every 10 days, every 11 days, every 12 days,every 13 days, every 2 weeks, every 3 weeks, every 4 weeks, or every 5weeks to a subject. In some embodiments, a dose of ASM described hereinis administered every 3 to 5 days, every 3 to 7 days, every 5 to 7 days,every 5 to 10 days, every 5 to 14 days, every 7 to 14 days, every 2 to 4weeks, or every 3 to 5 weeks to a subject. In certain embodiments, thefrequency of administration of a dose of ASM changes as the dose isadjusted. For example, a dose of 0.3 mg/kg may be administered everyweek or two weeks and a dose of 1 mg/kg may be administered every twoweeks or three weeks.

In some embodiments, a dose of ASM (e.g., a maintenance dose) isadministered to a subject for a period for 12 weeks, 20 weeks, 25 weeks,30 weeks, 35 weeks, 40 weeks, 45 weeks, 50 weeks, 52 weeks, 1 year, 1.5years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years or until thepatient experiences a related adverse event, a total bilirubin valuegreater than the bilirubin value for a human without ASMD (e.g., ahealthy human), a plasma ceramide concentration greater than the plasmaceramide concentration of a human without ASMD (e.g., a healthy human),or an acute phase response.

In certain embodiments, an initial low non-toxic dose of ASM isadministered every 3 days, every 4 days, every 5 days, every 6 days,every week, every 8 days, every 9 day, every 10 days, every 11 days,every 12 days, every 13 days, every 2 weeks, every 3 weeks, every 4weeks, or every 5 weeks to a subject. In some embodiments, a dose of ASMdescribed herein is administered every 3 to 5 days, every 3 to 7 days,every 5 to 7 days, every 5 to 10 days, every 5 to 14 days, every 7 to 14days, every 2 to 4 weeks, or every 3 to 5 weeks to a subject. In certainembodiments, the initial low non-toxic dose of ASM is administered for aperiod of 4 weeks, 6 weeks, 8 weeks, 12 weeks, 14 weeks or longer.

In certain embodiments, a successive increase in the dose of ASM isapproximately 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 1.2 mg/kg, 1.5 mg/kg,1.75 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg or 5 mg/kg higher than theprevious dose. In some embodiments, a successive increase in the dose ofASM 0.1 to 0.5 mg/kg, 0.1 mg/kg to 1 mg/kg, 0.5 mg/kg to 1 mg/kg, 0.5mg/kg to 2 mg/kg, 1 mg/kg to 2 mg/kg, 2 mg/kg to 4 mg/kg or 2 mg/kg to 5mg/kg higher than the previous dose.

Doses of ASM described herein can be administered by any route that isuseful in achieving a therapeutic effect. Specific routes ofadministration of doses of ASM include, but are not limited to,intravenous, intraventricular, intradermal, transdermal, subcutaneous,intramuscular, intranasal, inhalation, intrapulmonary, topical,transmucosal, intracranial, intrathecal, epidural and intra-synovial. Inone embodiment, a dose of ASM is administered systemically (e.g.,parenterally) to a subject in need thereof. In another embodiment, adose of ASM is administered locally to a subject in need thereof.

The dose of ASM enzyme that is effective in treating the somatic(non-central nervous system) manifestations of ASMD is not able toeffectively cross the blood-brain barrier. Thus, in specificembodiments, ASM is administered to a patient intraventicularly orintrathetically to the brain of a NPD patient. See, e.g., U.S. PatentApplication Publication No. 2009/0130079 and 2009/0123451, which areincorporated herein by reference in their entirety, for methods forintraventricular delivery of lysosomal storage enzymes to the brain. Incertain embodiments, the ASM is administered to a patientintracerebraventricularly. See, e.g., Dodge et al., 2009, ExperimentalNeurology 215: 349-357, which is incorporated herein by reference in itsentirety, for methods for intracerebraventricular infusion of ASM. Insome embodiments, the ASM is administered to a patient by indirectintraparenchymal injections. See, e.g., Yang et al., 2007, ExperimentalNeurology 207: 258-266. In some embodiments, a modified form of ASM thattargets the enzyme to the brain, such as described in Section 5.2,infra, is administered to a patient to treat ASMD.

Only a small percentage of the ASM enzyme administered to a patientreaches the lung. Thus, in specific embodiments, ASM is administered tothe lungs of a patient. In certain embodiments, ASM is administered byintranasal or inhalation to a patient. See, e.g., Ziegler et al., 2009,Molecular Genetics and Metabolism 97: 35-42, which is incorporatedherein by reference in its entirety, for information regarding theintranasal administration of ASM. In some embodiments, a modified formof ASM that targets the enzyme to the lung, such as described in Section5.2, infra, is administered to a patient to treat ASMD. In certainembodiments, the ASM is administered to a patient using a nebulizer.Delivery of ASM to the lungs may occur by intrapulmonary injectionthrough a bronchoscope, a metered-dose inhaler, or a nebulizer.

In certain embodiments, the ASM enzyme is administered systemically to apatient as well as locally administered to specific organs, such thebrain and lung. In some embodiments, the locally administration of theASM enzyme supplements the systemic administration of the enzyme. Inspecific embodiments, the ASM enzyme is administered locally to, e.g.,the brain or lungs, after the accumulated sphingomyelin in the patienthas been debulked by systemic administration (e.g., intravenousadministration).

In certain embodiments, a patient is genotyped prior to theadministration of ASM. In some embodiments, the glycosylation pattern ofthe ASM expressed by a patient is determined prior to administration ofASM. The administration of ASM that is similar/compatible with thatendogenously expressed by a patient may reduce the potential forimmunogenicity.

In certain embodiments, the activity of endogenously expressed ASM isdetermined prior to the administration of ASM. The activity ofendogenously expressed ASM can be measured in DBS and in culturedfibroblasts using techniques known to one of skill in the art.

In a specific embodiment, the methods for treating ASMD provided hereinreduce spleen volume as assessed by techniques known in the art, e.g.,MRI. In another specific embodiment, the methods for treating ASMDprovided herein reduce liver sphingomyelin levels as assessed bytechniques known in the art, e.g., biochemical analysis and/orhistomorphometric analysis of liver samples. In another specificembodiment, the methods for treating ASMD provided herein increaseexercise capacity as assessed by techniques known in the art, e.g.,maximum workload by cycle erogmetry, including percent predicted maximumworkload, peak oxygen consumption, and carbon dioxide production. Inanother specific embodiment, the methods for treating ASMD providedherein increase in pulmonary function as assessed by techniques known inthe art, e.g., DLco, FVC, FEV, and/or TLC. In another specificembodiment, the methods for treating ASMD provided herein decrease inbronchial alveolar lavage (BAL) sphinomyelin. In another specificembodiment, the methods for treating ASMD provided herein decrease inliver volume as assessed by techniques known in the art, e.g., Mill. Inanother specific embodiment, the methods for treating ASMD providedherein improve lung appearance as assessed by techniques known in theart, e.g., high resolution CT scan or chest X-ray. In another specificembodiment, the methods for treating ASMD provided herein improve lungclearance. In another specific embodiment, the methods for treating ASMDprovided herein decrease in sphinomyelin concentration in the liver,skin, plasma and DBS. In another specific embodiment, the methods fortreating ASMD provided herein reduce serum chitotriosidase levels. Inanother specific embodiment, the methods for treating ASMD providedherein reduce serum CCL18 levels. In another specific embodiment, themethods for treating ASMD provided herein improve a patient's lipidprofile (e.g., decrease cholesterol). In another specific embodiment,the methods for treating ASMD provided herein reduce or ameliorate theseverity of ASMD and/or a symptom associated therewith. In anotherspecific embodiment, the methods for treating ASMD provided hereinreduce the duration of a symptom associated with ASMD. In anotherspecific embodiment, the methods for treating ASMD provided hereinprevent the recurrence of a symptom associated with ASMD. In anotherspecific embodiment, the methods for treating ASMD provided herein areduces hospitalization of a subject. In another specific embodiment,the methods for treating ASMD provided herein reduces hospitalizationlength. In another specific embodiment, the methods for treating ASMDprovided herein increases the survival of a subject. In another specificembodiment, the methods for treating ASMD provided herein reduces themortality of subject. In another specific embodiment, the methods fortreating ASMD provided herein decreases hospitalization rate of asubject. In another specific embodiment, the methods for treating ASMDprovided herein reduces the number of symptoms associated with ASMD. Inanother specific embodiment, the methods for treating ASMD providedherein increases symptom-free survival of ASMD patients. In anotherspecific embodiment, the methods for treating ASMD provided hereinimprove in neurological function (e.g., psychomotor function, socialresponsiveness, etc.) of a subject.

In another specific embodiment, the methods for treating ASMD providedherein improve a patient's quality of life. In certain specificembodiments, the method for treating ASMD provided herein improve apatient's quality of life as assessed by, e.g., the Brief FatigueInventory (BFI; Mendoza et al., 1999, Cancer 85(5): 1186-1196), theBrief Pain Inventory-Short Form (BPI-SF; Cleeland et al., 1994, Ann AcadMed Singapore 23(2): 129-138), the ASM-Health Assessment Questionnaire,which is a composite of the BFI, BPI-SF, and Short Form-36 HealthSurvey, the Chronic Respiratory Disease Questionnaire Self-AdministeredStandardized (CRQ-SAS; Schunemann et al., 2005, Eur. Respir. J. 25:31-40) to assess dyspnea and fatigue, and the Activity Measure forPost-Acute Care (AM-PAC), a computer-adaptive test that assessesfunctional motilities (e.g., mobility, self-care, and appliedcognition). Acid spHingomyelinase enzyme

ASM refers to any form the acid sphinomyelinase enzyme that retains theability to hydrolyze sphingomyelin to ceramide and phosphorylcholine asassessed by techniques known to one of skill in the art, such as thosedescribed in U.S. Pat. Nos. 4,039,388, 4,082,781, 5,686,240, and7,563,591, and International Publication Nos. WO 2007/078806 and WO2006/058385, which are incorporated herein by reference in theirentirety. In a specific embodiment, an acid sphingomyelinease enzyme hasthe ability to hydrolyze sphingomyelin to ceramide and phosphorylcholinein the fluorescence-based, high-performance liquid chromatographic assaydescribed in He et al., 2003, Analytical Biochemistry 314: 116-120. In aspecific embodiment, an acid sphingomyelinase has at least 30%, 35%,40%, 45%, 50%, 75%, 80%, 85%, 80%, 90%, 95% or 98%, or 30% to 50%, 40%to 50%, 50% to 75%, 50% to 90%, 75% to 80%, 75% to 90%, 75% to 95%, or85% to 95% of the activity of ASM-1 (SEQ ID NO: 1, infra) as measuredby, e.g., the assay described in He et al., 2003, AnalyticalBiochemistry 314: 116-120.

In a specific embodiment, the ASM is a human ASM. There are variousisoforms of human ASM resulting from alternative splicing. One of thehuman ASM isoforms, human ASM isoform 1 (sometimes referred to asASM-1), has the amino acid sequence found at UniProtKB/Swiss-ProtAccession No. P17405-1. Another human ASM isoform, human isoform 2(sometimes referred to as ASM-2), has the amino acid sequence found atUniProtKB/Swiss-Prot. Accession No. P17405-2. A third human ASM isoform,human isoform 3 (sometimes referred to as ASM-3), has the amino acidsequence found at UniProtKB/Swiss-Prot. Accession No. P17405-3. Theamino acid sequence of ASM-1, which is the most abundant isoform, isprovided below:

        10         20         30         40MPRYGASLRQ SCPRSGREQG QDGTAGAPGL LWMGLVLALA        50         60         70         80LALALALSDS RVLWAPAEAH PLSPQGHPAR LHRIVPRLRD        90        100        110        120VFGWGNLTCP ICKGLFTAIN LGLKKEPNVA RVGSVAIKLC       130        140        150        160NLLKIAPPAV CQSIVHLFED DMVEVWRRSV LSPSEACGLL       170        180        190        200 LGSTCGHWDI FSSWNISLPT VPKPPPKPPS PPAPGAPVSR       210        220        230        240ILFLTDLHWD HDYLEGTDPD CADPLCCRRG SGLPPASRPG       250        260        270        280AGYWGEYSKC DLPLRTLESL LSGLGPAGPF DMVYWTGDIP       290        300        310        320AHDVWHQTRQ DQLRALTIVT ALVRKFLGPV PVYPAVGNHE       330        340        350        360STPVNSFPPP FIEGNHSSRW LYEAMAKAWE PWLPAEALRT       370        380        390        400LRIGGFYALS PYPGLRLISL NMNFCSRENF WLLINSTDPA       410        420        430        440GQLQWLVGEL QAAEDRGDKV HIIGHIPPGH CLKSWSWNYY       450        460        470        480RIVARYENTL AAQFFGHTHV DEFEVFYDEE TLSRPLAVAF       490        500        510        520LAPSATTYIG LNPGYRVYQI DGNYSGSSHV VLDHETYILN       530        540        550        560LTQANIPGAI PHWQLLYRAR ETYGLPNTLP TAWHNLVYRM       570        580        590        600RGDMQLFQTF WFLYHKGHPP SEPCGTPCRL ATLCAQLSAR        610        620ADSPALCRHL MPDGSLPEAQ SLWPRPLFC(SEQ ID NO:1). ASM-2 differs from ASM-1 in that amino acid residues 363to 374 of ASM-1 (i.e., IGGFYALSPYPG (SEQ ID NO:2)) are replaced withamino acids YLSSVETQEGKR (SEQ ID NO:3) and amino acid residues 375 to418 of ASM-1 are missing from ASM-2. ASM-3 differs from ASM-1 in thatamino acid residues 363 to 418 of ASM-1 are missing from ASM-3. To theextent that ASM-2 and ASM-3 have enzymatic activity as measured by,e.g., the assay described in He et al., 2003, Analytical Biochemistry314: 116-120, they are included as an ASM that could be administered toa subject. In a specific embodiment, to the extent that ASM-2 and ASM-3have at least 30%, 35%, 40%, 45%, 50%, 75%, 80%, 85%, 80%, 90%, 95% or98%, or 30% to 50%, 40% to 50%, 50% to 75%, 50% to 90%, 75% to 80%, 75%to 90%, 75% to 95%, or 85% to 95% of the activity of ASM-1 as measuredby, e.g., the assay described in He et al., 2003, AnalyticalBiochemistry 314: 116-120, they are included as an ASM that could beadministered to a subject.

In a specific embodiment, the human ASM has the amino acid sequence ofhuman acid sphingomyelinase disclosed in SEQ ID NO:1, supra, the aminoacid sequence of a human acid sphingomyelinase disclosed in U.S. Pat.No. 6,541,218 (e.g., SEQ ID NO:2 in U.S. Pat. No. 6,541,218), or theamino acid sequence disclosed in FIG. 3 of Schuchman et al., 1991, J.Biol. Chem. 266: 8531-8539, each of which is incorporated herein byreference in its entirety.

In specific embodiments, the human ASM is the processed mature form. Inother embodiments, the human ASM is the immature, unprocessed form. Withrespect to ASM-1, the immature form is 629 amino acids in length andcontains the signal peptide found at amino acid residues 1 to 46. Themature form of ASM-1 lacks that signal peptide and is from amino acidresidue 47 to amino acid residue 629. Human ASM-1 contains a saposinB-type domain from amino acid residues 85 to 169. With respect to ASM-1,the following amino acid residues are glycosylated (in particular,N-linked glycosylated): 86, 175, 335, 395, and 520.

In addition to the isoforms of human ASM, there are various naturallyoccurring variants of human ASM. For example, naturally occurringvariants of the human ASM gene with different numbers of hexanucleotiderepeat units occurring within the region of the gene encoding theputative signal peptide of ASM have been identified. See, e.g., Wan andSchuchman, 1995, Biochimica et Biophysica Acta 1270: 207-210 (which isincorporated herein by reference in its entirety) which the describesthe identification of five alleles corresponding to nine, seven, six,five and four hexanucleotide repeats. Further, naturally occurringvariants of human ASM with single amino acid variations have beenidentified. See, e.g., Schuchman et al., 1991, J. of Biol. Chem 266:8531-8539 and Schuchman et al., 1991, Nucleic Acids Research 19(11):3160, which are incorporated herein by reference in their entirety, forinformation regarding single polymorphisms. To the extent that any ofthe naturally occurring variants of human ASM have enzymatic activity asmeasured by, e.g., the assay described in He et al., 2003, AnalyticalBiochemistry 314: 116-120, they are included as an ASM that could beadministered to a subject. In a specific embodiment, to the extent thatany of the naturally occurring variants of human ASM have at least 30%,35%, 40%, 45%, 50%, 75%, 80%, 85%, 80%, 90%, 95% or 98%, or 30% to 50%,40% to 50%, 50% to 75%, 50% to 90%, 75% to 80%, 75% to 90%, 75% to 95%,or 85% to 95% of the activity of ASM-1 as measured by, e.g., the assaydescribed in He et al., 2003, Analytical Biochemistry 314: 116-120, theyare included as an ASM that could be administered to a subject.

Many single nucleotide polymorphisms (SNPs) for the gene encoding humanASM have been identified (see, e.g., the ncbi website:ncbi.nlm.nih.gov/projects/SNP/for examples of SNPs). To the extent thatany of the SNPs in the gene encode of an ASM that has enzymatic activityas measured by, e.g., the assay described in He et al., 2003, AnalyticalBiochemistry 314: 116-120, they are included as an ASM that could beadministered to a subject. In a specific embodiment, to the extent thatany of the SNPs in the gene encode of an ASM that has at least 30%, 35%,40%, 45%, 50%, 75%, 80%, 85%, 80%, 90%, 95% or 98%, or 30% to 50%, 40%to 50%, 50% to 75%, 50% to 90%, 75% to 80%, 75% to 90%, 75% to 95%, or85% to 95% of the activity of ASM-1 as measured by, e.g., the assaydescribed in He et al., 2003, Analytical Biochemistry 314: 116-120, theyare included as an ASM that could be administered to a subject. Incertain embodiments, the ASM is a modified form of human ASM. In aspecific embodiment, the modified form of human ASM is one disclosed inU.S. Pat. No. 7,527,956, which is incorporated herein by reference inits entirety. In another embodiment, the modified form of human ASM isone disclosed in International Publication No. WO 2008/136451, which isincorporated herein by reference in its entirety. In some specificembodiments, the ASM is a modified form of human ASM that has one, twoor more of the following properties: (i) increased targeting to sites ofpathology (e.g., the lung and/or brain) relative to unmodified humanASM, (ii) increased stability relative to unmodified human ASM, and(iii) increased activity relative to unmodified human ASM. Techniquesknown in the art can be used to measure the stability, activity andtargeting of ASM. In a specific embodiment, the techniques described inGamacho et al., 2008, J. Pharmacol. Exp. Ther. 325: 400-408 (which isincorporated herein by reference in its entirety) for assessingtargeting of ASM are used. In another specific embodiment, thetechniques described in He et al., 1999, Biochimica et Biophysica Acta1432: 251-264 or Dhami et al., 2001, Lab. Invest. 81: 987-999 (which areincorporated herein by reference in their entirety) for assessingstability of the ASM are used. In another specific embodiment, thetechniques described in He et al., 2003, Analytical Biochemistry 314:116-120 (which is incorporated herein by reference in its entirety) forassessing ASM activity are used.

In one embodiment, the ASM is a modified form of human ASM that hasincreased enzymatic activity relative to unmodified human ASM, such asASM-1. See, e.g., Qiu et al., 2003, J. Biol. Chem. 278(35): 32744-32752and U.S. Pat. No. 7,527,956 for a mutant forms of recombinant human ASMwith enzymatic activity with higher activity than wild-type recombinanthuman ASM (such as, e.g., ASM-1). In some embodiments, the ASM hasincreased enzymatic activity relative to unmodified human ASM (such as,e.g., ASM-1) due to the addition of zinc cations. See, e.g., Schissel etal., 1998, J. Biol. Chem 273: 18250-18259, which is incorporated hereinby reference in its entirety, for a discussion regarding the role ofzinc in ASM activity. In another embodiment, the ASM is a modified formof human ASM that has increased affinity for a natural receptor of humanASM (e.g., mannose 6-phosphate or high mannose) relative to unmodifiedhuman ASM (such as, e.g., ASM-1). In a specific embodiment, the ASM isconjugated to an oligosaccharide, such as described in U.S. Pat. No.7,001,994 and International Patent Application Publication No. WO2010/075010 and United States Patent Application Publication No.2010/0173385 (which are incorporated herein by reference in theirentirety), to increase the targeting the enzyme for its naturalreceptor. In another embodiment, the ASM is a modified form of human ASMthat binds to an alterative receptor (e.g., intercellular adhesionmolecule (ICAM)-1 which may increase targeting to organs such as thelung) than the natural receptor for ASM. In embodiment, the ASM is amodified form of human ASM that has increased stability relative tounmodified human ASM (such as, e.g., ASM-1).

In some embodiments, the ASM is conjugated or fused, directly orindirectly, to a targeting moiety, such as insulin-like growth factor(IGF)-I, IGF-II, leptin, granulocyte colony stimulating factor (G-CSF),or a humanized antibody that binds to human insulin receptor (HIR). See,e.g., U.S. Patent Application Publication No. 2009/0029467 and U.S. Pat.No. 7,560,424 (which is incorporated herein by reference in itsentirety) for a discussion of the use of IGF-I as a targeting moiety forlysosomal storage enzymes, and U.S. Pat. No. 7,396,811 (which isincorporated herein by reference by reference in its entirety) for adiscussion of the use of IGF-II as a targeting moiety for lysosomalstorage enzymes. The targeting moieties IGF-I and IGF-II can facilitatetargeting the ASM enzyme to the lyosome. For a discussion of the use ofhormones, such as G-CSF and leptin as targeting moieties see, e.g.,International Patent Application Publication No. WO 2007/091250 and U.S.Patent Application Publication No. 2010/0183577, which are incorporatedherein by reference herein by reference in their entirety. See, e.g.,U.S. Patent Application Publication No. 2004/0101904, which isincorporated herein by reference in its entirety, for a discussion of ahumanized monoclonal antibody that binds human insulin receptor (HIR)linked to a pharmaceutical agent, such as an enzyme. The targetingmoieties G-CSF, leptin and an antibody that binds to HIR can facilitatetargeting of the ASM enzyme to the brain.

In certain embodiments, the ASM is a modified form of ASM (e.g., amodified form of human ASM) with a decrease in activity relative tounmodified ASM (such as, an unmodified human ASM, e.g., ASM-1). In someembodiments, the ASM is a modified form of ASM (e.g., a modified form ofhuman ASM) with an increase in targeting to sites of pathology and adecrease in enzymatic activity relative to unmodified ASM (such as anunmodified human ASM, e.g., ASM-1).

In certain embodiments, the ASM is a highly phosphorylated form of ASM.See, e.g., U.S. Patent Application Publication No. 2002/0150981, whichis incorporated herein by reference, for a discussion of techniques forproducing highly phosphorylated forms of lysosomal enzymes.

The ASM enzyme can be produced by any method known in the art, includingbut not limited to recombinant DNA methods, cDNA cloning (see, e.g.,Schuchman et al., 1991, J. Biol. Chem 266: 8531-8539 and U.S. Pat. No.5,773,278, which are incorporated herein by reference in their entiretyfor cDNA clones of human ASM), genomic cloning, gene activation (see,e.g., U.S. Pat. No. 5,641,670, which is incorporated herein by referencein its entirety for gene activation techniques), or selected cell lines(e.g., mammalian, yeast, prokaryotic, insect cells (e.g., Sf 9, Sf21,Trichoplusia ni, Spodoptera frugiperda and Bombyx mori), and plantcells) that produce high levels of the ASM enzyme. ASM as well asmethods for producing ASM are described, e.g., in U.S. Pat. Nos.5,773,278; 6,541,218; 5,686,240; and 7,527,956; each of which isincorporated herein by reference in its entirety.

In a specific embodiment, the ASM is a recombinantly produced ASM (e.g.,a recombinantly produced human ASM). Cell expression systems whichpossess the cellular machinery and elements for the proper processing,i.e., signal cleavage, glycosylation, phosphorylation and proteinsorting are preferred. For example, mammalian cell expression systemsare preferred for the expression of biologically active enzymes that areproperly folded and processed; when administered in humans suchexpression products should exhibit proper tissue targeting and noadverse immunological reaction.

In certain embodiments, the ASM (e.g., human ASM) is produced byoverexpression of ASM cDNA in mammalian cells. In a specific embodiment,the human ASM is produced by overexpression of human ASM cDNA in Chinesehamster ovary (CHO) cells. See, e.g., He et al., 1999, Biochimia etBiophsyica Acta 1432: 251-264 for methods for overexpression of humanASM cDNA in CHO cells, and U.S. Pat. No. 6,541,218 each of which isincorporated by reference herein in its entirety. In certainembodiments, the human ASM is produced by overexpression of human ASMcDNA in CHO cells that co-express alpha-2,6-sialytransferase. See, e.g.,U.S. Pat. No. 5,047,335, incorporated herein by reference in itsentirety, which describes CHO cells engineered to express alpha2,6-sialytransferase.

In another specific embodiment, the mammalian cells are human cells.Examples of human cells that may be used to recombinantly express ASM(e.g., human ASM) include, but are not limited to Crucell Per.C6 cells,HT 1080 cells, HeLa cells, HEK 293 cells, 293T cells, WI38 cells, HuT292cells, LF 1043 (HEL) cells, MRC-5 cells, TMK-1 cells, BT483 cells,Hs578T cells, HTB2 cells, HTB3 cells, HTB4 cells, BT 20 cells, T47Dcells. CRL7O3O cells, HsS78Bst cells, 721 cells, A2780 cells, A172cells, A253 cells, COR-L23/R23 cells, COV-434 cells, DU145 cells, DuCaPcells, EM2 cells, Saos-2 cells, U373 cells, WM39 cells, L132 cells,A-5489 cells, G-293 cells, G-401 cells, CAKI-1 cells, RD cells, and YARcells. Other exemplary human cells that may be used to express ASMinclude those human cell lines listed in the Immuno PolymorphismDatabase of the European Molecular Biology Laboratory, and the databaseof the U.S. National Institutes of Health, as well as those commerciallyavailable form, for example, the American Type Culture Collection,Manassas, Va., and Lifeline Cell Technology, Walkersville, Md. Whenusing human cell lines for production, rhASM can be produced usingcDNAs, gDNAs for hASM or by gene activation techniques such as thosedescribed in U.S. Pat. No. 5,641,670 which is incorporated by referenceherein in its entirety.

Examples of other mammalian cells that may be used to produce ASM (e.g.,human ASM) include, but are not limited to, Vero cells, VERY cells, BHKcells, COS cells, MDCK cells, or 3T3 cells. In certain embodiments,myeloma cells are used to produce ASM (e.g., human ASM). Non-limitingexamples of myeloma cells include NS0 cells, 45.6 TG1.7 cells, AF-2clone 9B5 cells, AF-2 clone 9B5 cells, J558L cells, MOPC 315 cells,MPC-11 cells, NCI-H929 cells, NP cells, NS0/1 cells, P3 NS1 Ag4 cells,P3/NS1/1-Ag4-1 cells, P3U1 cells, P3X63Ag8 cells, P3X63Ag8.653 cells,P3X63Ag8U.1 cells, RPMI 8226 cells, Sp20-Ag14 cells, U266B1 cells,X63AG8.653 cells, Y3.Ag.1.2.3 cells, and YO cells.

In some embodiments, a plant cell culture system is used for expressionof the ASM. See, e.g., U.S. Pat. Nos. 5,929,304; 7,504,560; 6,770,799;6,551,820; 6,136,320; 6,034,298; 5,914,935; 5,612,487; and 5,484,719,U.S. patent application publication Nos. 2009/0208477, 2009/0082548,2009/0053762, 2008/0038232, 2007/0275014 and 2006/0204487, and Shoji etal., 2008, Vaccine, 26(23):2930-2934, and D'Aoust et al., 2008, J. PlantBiotechnology, 6(9):930-940 (which are incorporated herein by referencein their entirety) for plant cells and methods for the production ofproteins utilizing plant cell culture systems. In particular, U.S.patent application Nos. 2009/0208477, 2008/0038232 and 2006/0204487describe the expression and production of enzymatically active highmannose lysosomal enzymes using transgenic plant root, particularlycarrot cells. In a specific embodiment, carrot cells are engineered toexpress ASM. In certain embodiments, algae (e.g., Chlamydomonasreinhardtii) may be engineered to express ASM (see, e.g., Rasala et al.,2010, Plant Biotechnology Journal (Published online Mar. 7, 2010, whichis incorporated herein by reference in its entirety).

Tissue and cellular uptake of ASM is mediated by both the high mannoseresidues (e.g., in macrophages) and by mannose-6-phosphate (e.g., inliver). Thus, production of ASM with altered glycan content and/orphosphorylation may be desired to enhance drug distribution. Forexample, ASM can be produced by cells having a mutation, e.g., aknockout for, at least one Golgi processing mannosidase. In oneembodiment, the mutation reduces the expression of the gene, reducesprotein or activity levels, or alters the distribution or other posttranslational modifications of the mannosidase, e.g., the processing ofthe carbohydrate chains. In a specific embodiment, the mutation reducesthe level of the Golgi processing mannosidase activity. The mutation canbe in a class 1 processing mannosidase; a class 2 processingmannosidase; a class 1 processing mannosidase and a class 2 processingmannosidase. Class 1 processing mannosidase includes: Golgi mannosidaseIA; Golgi mannosidase IB; Golgi mannosidase IC. Class 2 processingmannosidase includes: Golgi mannosidase II. See, e.g., InternationalPatent Application Publication No. WO 02/15927 and U.S. Pat. No.7,138,262, which are incorporated herein by reference in their entirety,for methods of producing a high mannose protein.

In certain embodiments, cells expressing ASM are cultured in thepresence of mannosidase inhibitor, such as an antibody, kifunensine,swainsonine, mannostatin, 6-deoxy-1, 4-dideoxy-1,4-imino-D-mannitol(6-deoxy-DIM), or 6-deoxy-6-fluoro-1, 4-dideoxy-1, 4-imino-D-mannitol(6-deoxy-6-fluoro-DIM). The culture of ASM in the presence of suchinhibitors may result in the production of a high mannose ASM. In someembodiments, the cells expressing ASM include a nucleic acid sequence,such as an antisense molecule or ribozyme, which can bind to orinactivate a cellular mannosidase nucleic acid sequence, e.g., mRNA, andinhibit expression of the protein. See, e.g., International PatentApplication Publication No. WO 02/15927 and U.S. Pat. No. 7,138,262,which are incorporated herein by reference in their entirety, formethods of producing a high mannose protein.

In certain embodiments, the carbohydrate chains of the ASM enzymerecombinantly expressed are remodeled by sequential treatment withvarious enzymes, such as neuraminidase, galactosidase and .beta.-Nacetylglucosaminidase. See, e.g., U.S. Pat. No. 5,549,892, which isincorporated herein by reference in its entirety, for a methods forremodeling carbohydrate chains of a lysosomal enzyme.

Uptake of ASM, mediated by mannose-6-phosphate (M6P) can be enhanced bymodification of ASM to produce highly phosphorylated mannose residuesand M6P. For example, ASM can be modified by recombinant technology tointroduce additional mannose-6-phosphate to the ASM for enhancingcellular uptake. See, e.g., Matsuoka et al., 2010 Mole. Ther.18:1519-1526 which is incorporated herein by reference in its entirety.In other embodiments, ASM can be coupled to highly phosphorylatedoligosaccharide derivatives containing mannose 6-phosphate (M6P). See,e.g., U.S. Pat. No. 7,001,994, U.S Patent Application PublicationUS2010/0173385 and International Publication No. WO2010/075010. Inanother approach, a yeast culture system can be used for the expressionof recombinant ASM that contains additional highly phosphorylatedmannose-6-phosphate residues. See, e.g., Akeboshi et al., 2009Glycobiology 19(9):1002-1009 which is incorporated by reference hereinin its entirety.

Once ASM has been produced, it may be isolated or purified by any methodknown in the art for isolation or purification of a protein, forexample, by chromatography (e.g., ion exchange, high performance liquidchromatography, affinity, particularly by affinity for ASM, by ProteinA, and sizing column chromatography), centrifugation, differentialsolubility, or by any other standard technique for the isolation orpurification of proteins. Where the ASM enzyme is secreted by thecultured cells, ASM may be readily recovered from the culture medium.See, e.g., He et al., 1999, Biochimia et Biophsyica Acta 1432: 251-264for methods for purifying ASM.

The ASM enzyme can be formulated for any route of administration (e.g.,infusion, subcutaneous, intramuscular, intrathecal, intraventricular,intranasal, inhalation or intradermal). The ASM enzyme can be suppliedin a lyophilized form that is reconstituted before use with, e.g.,sterile saline (e.g., 0.9% sodium chloride) or sterile water.Alternatively, the ASM enzyme can be supplied in an aqueous form. Incertain embodiments, ASM is administered to a subject in a formulationcomprising zinc. In some embodiments, the ASM enzyme is administered tothe patient by infusion using, e.g., a syringe pump or an infusion bagwith a pump.

In certain embodiments, ASM is administered to a subject in a carrier,such as liposomes or a polycationic carrier. See, e.g., U.S. Pat. No.5,716,614, which is incorporated herein by reference in its entirety,for a carriers that can be used to administered ASM. In someembodiments, the ASM administered to a subject in ICAM-1-targetnanocarriers. See, e.g., Muro et al., 2006, Mol. Ther. 13(1): 135-141,which is incorporated herein by reference, for ASM delivery usingICAM-1-targeted nanocarriers.

5.2. Patient Populations

In specific embodiments, a subject treated for ASMD in accordance withthe methods provided herein is a human who has or is diagnosed as havingone or more mutations in the gene encoding acid sphingomyelinase. Inparticular embodiments, a subject treated for ASMD in accordance withthe methods provided herein is a human who has or is diagnosed as havingNiemann-Pick disease (NPD). In one embodiment, a subject treated forASMD in accordance with the methods provided herein is a human who hasor is diagnosed as having NPD Type A. In another specific embodiment, asubject treated for ASMD in accordance with the methods provided hereinis a human who has or is diagnosed as having NPD Type B.

In certain embodiments, a subject treated for ASMD in accordance withthe methods provided herein has one or more mutations in the SMPD1 gene.In some embodiments, the mutation is a missense mutation. In otherembodiments, the mutation is a deletion that results is the deletion ofone, two or more amino acid residues. In specific embodiments, a subjecttreated for ASMD in accordance with the methods provided herein has oneor more of the mutations

TABLE 1 Amino Acid Position of ASM-1 Mutation  49 D → V in NPDB  92 C →W in NPDB 103 L → P in NPDA and NPDB 130 V → A in NPDB 137 L → P in NPDB157 C → R in NPDB 166 G → R in NPDB 176 I → N in NPDB 184 P → L inNPDA/NPDB 196 A → P in NPDB 200 R → C in NPDB 225 L → M in NPDB 225 L →P in NPDB 228 R → C in NPDB 228 R → H in NPDA/NPDB 232 G → D in NPDB 241A → V in NPDA/NPDB 242 G → R in NPDB 244 W → C in NPDB 245 G → S in NPDAand NPDB 246 E → K in NPDA 246 E → Q in NPDA 248 S → R in NPDA and NPDB251 D → E in NPDA/NPDB 278 D → A in NPDA/NPDB 281 A → T in NPDB 289 R →H in NPDB 292 Q → K in NPDA/NPDB 294 R → Q in NPDA 302 L → P in NPDA 313Y → H in NPDA 319 H → Y in NPDA 323 P → A in NPDB. 330 P → R in NPDB 341L → P in NPDA/NPDB 357 A → D in NPDB 367 Y → C in NPDA 371 P → S in NPDB376 R → H in NPDB 376 R → L in NPDB 379 S → P in NPDB 382 M → I in NPDA383 N → S in NPDB 389 N → T in NPDA 390 Missing in NPDA. 391 W → G inNPDB 413 A → V in NPDB 421 H → R in NPDA 421 H → Y in NPDB 431 C → R inNPDB 432 L → P in NPDB 435 W → C in NPDB 436 S → R in NPDB 446 Y → C inNPDA 450 L → P in NPDA 452 A → V in NPDB 456 G → D in NPDB 463 F → S inNPDA 467 Y → S in NPDA 474 R → W in NPDB 475 P → L in NPDA and NPDB 480F → L in NPDB 482 A → E in NPDA 485 A → V in NPDB 486 T → A in NPDB 488Y → N in NPDB 494 G → S in NPDB 496 R → C in NPDB 496 R → H in NPDA 496R → L in NPDA 514 H → Q in NPDB 515 E → V in NPDB 517 Y → C in NPDA 533W → R in NPDB 537 Y → H in NPDA 549 L → P in NPDB 563 D → Y in NPDB 576K → N in NPDB 577 G → S in NPDA 592 Missing in NPDA. 600 R → H in NPDB600 R → P in NPDB 608 Missing in NPDBSee, e.g., Simonaro et al., 2002, Am. J. Hum. Genet. 71: 1413-1419,which is incorporated herein by reference, for mutations in the acidsphingomyelinase gene (designated SMPD1).

In certain embodiments, a subject treated for ASMD in accordance withthe methods provided herein endogenously expresses ASM with 2 to 5%, 5to 10%, 5 to 15%, 5 to 20%, 5% to 30%, or 20% to 30% of the activity ofnormal, human ASM, e.g., ASM-1. In some embodiments, a subject treatedfor ASMD in accordance with the methods provided herein endogenouslyexpresses ASM with less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2% or1% of the activity of normal, human ASM, e.g., ASM-1. See, e.g., U.S.Pat. Nos. 4,039,388, 4,082,781, 5,686,240, and 7,563,591, andInternational Publication Nos. WO 2007/078806 and WO 2006/058385, whichare incorporated herein by reference in their entirety, for techniquesthat can be used to measure the activity of ASM. In a specificembodiment, the fluorescence-based, high-performance liquidchromatographic assay described in He et al., 2003, AnalyticalBiochemistry 314: 116-120 (which is incorporated herein by reference) isused to measure the activity of ASM.

In certain embodiments, a subject treated for ASMD in accordance withthe methods provided herein displays one or more symptoms of NPD.Symptoms of NPD include, but are not limited to, distended abdomens,hepatomegaly, splenomegly, hepatosplenomegaly, neutropenia, pulmonarydisease, lymphoadenopathy, the presence of histochemicallycharacteristic NPD foam cells, anemia (e.g., microcytic anemia),thrombocytopenia, recurrent vomiting, chronic constipation, growthfailure (e.g., decreased liner growth and body weight), delayed puberty,recurrent bruising, recurrent bleeding, atherogenic lipid profile (highcholesterol, triglycerides, LDL, and low HDL), pain (headache, back,extremities, abdomen), fatigue, early satiety, low endurance,osteopenia, neurological manifestations, and respiratory difficulties(e.g., interstitial lung disease, shortness of breath). Neurologicalmanifestation of NPD include cherry red spot, hypotonia, muscleweakness, psychomotor retardation, spasticity, social unresponsiveness,irritability, and seizures.

In certain embodiments, a subject treated for ASMD in accordance withthe methods provided herein is a human subject that displays two or moreclinical features consistent with non-neuropathic NPD. In specificembodiments, a subject treated for ASM in accordance with the methodsprovided herein is a human subject that displays two or more clinicalfeatures consistent with non-neuropathic NPD: thrombocytopenia, anemia,neutropenia, hepatomegaly, splenomegaly, and pulmonary disease. In otherembodiments, a subject treated for ASMD in accordance with the methodsprovided herein is a human subject that displays two or more clinicalfeatures consistent with neuropathic NPD.

In certain embodiments, a subject treated for ASMD in accordance withthe methods provided herein is a human with a spleen volume two, three,four, five, six, seven, eight, nine, ten, eleven or twelve times greaterthan the spleen volume of a healthy human as assessed by techniquesknown in the art, such as, e.g., magnetic resonance imaging (MM). Inspecific embodiments, a subject treated for ASMD in accordance with themethods provided herein is a human with a spleen volume greater thaneight times the spleen volume of a healthy human as assessed bytechniques known in the art, such as, e.g., MRI. In some embodiments, asubject treated for ASMD in accordance with the methods provided hereinis a human with a spleen volume eight to twelve, nine to twelve, ten totwelve, or twelve to fourteen times greater than the spleen volume of ahealthy human as assessed by techniques known in the art, such as, e.g.,MRI. In specific embodiments, a subject treated for ASMD in accordancewith the methods provided herein is a human with a spleen volume ≥8multiples of normal (MN) (i.e., 1.6% of body weight) as assessed bytechniques known in the art, such as, e.g., MRI.

In certain embodiments, a subject treated for ASMD in accordance withthe methods provided herein is a human with a diffusing capacity(DL_(CO)) between 20% to 80%, 25% to 80%, 30% to 80%, 40% to 80%, 50% to80%, or 60% to 80% of the predicted DL_(CO) of a healthy human. In someembodiments, a subject treated for ASMD in accordance with the methodsprovided herein is a human with a DL_(CO) of between 20% to 90%, 25% to90%, 30% to 90%, 40% to 90%, 50% to 90%, 60% to 90%, or 70% to 90% ofthe predicted DL_(CO) of a healthy human. DL_(CO) measures the rate ofdiffusion of a diffusion-limited gas (e.g., carbon monoxide) per minuteacross the alveolocapillary membrane. DL_(CO) can be calculated bycomparing the amount of carbon monoxide exhaled following a known amountof inhaled carbon monoxide. Techniques known in the art can be used toassess DL_(CO).

In certain embodiments, a subject treated for ASMD in accordance withthe methods provided herein is a human infant. In some embodiments, asubject treated for ASMD in accordance with the methods provided hereinis a human that is 2 to 3 months old, 2 to 6 months old, 3 to 6 monthsold, 4 to 6 months old, 5 to 8 months old, or 6 to 9 months old. Inother embodiments, a subject treated for ASMD in accordance with themethods provided herein is a human child. In some embodiments, a subjecttreated for ASMD in accordance with the methods provided herein is ahuman that is 1 to 3 years old, 2 to 3 years old, 3 to 5 years old, 4 to5 years old, 5 to 7 years old, or 6 to 9 years old.

In certain embodiments, a subject treated for ASMD in accordance withthe methods provided herein is a human adult. In other embodiments, asubject treated for ASMD in accordance with the methods described hereinis a elderly human. In some embodiments, a subject treated for ASMD inaccordance with the methods provided herein is a human that is 10 to 18years old, 10 to 20 years old, 12 to 20 years old, 15 to 20 years old,20 to 25 years old, 21 to 25 years old, 21 to 30 years old, 25 to 30years old, 30 to 35 years old, 35 to 40 years old, 40 to 45 years old,45 to 50 years old, 50 to 55 years old, 55 to 60 years old, 60 to 65years old, 65 to 70 years old, 70 to 75 years old, 75 to 80 years old,80 to 85 years old, 85 to 90 years old, 90 to 95 years old or 95 to 100years old.

In specific embodiments, a subject treated for ASMD in accordance withthe methods provided herein is a human female. In other embodiments, asubject treated for ASMD in accordance with the methods provided hereinis a human male. In certain embodiments, a subject treated for ASMD inaccordance with the methods provided herein is a female human that isnot pregnant or is not breastfeeding. In other embodiments, a subjecttreated for ASMD in accordance with the methods provided herein is afemale human that is pregnant or will become pregnant, or isbreastfeeding.

In certain embodiments, a subject treated for ASMD in accordance withthe methods provided herein is a human that has not had a major organtransplant (e.g., liver or bone marrow transplant). In otherembodiments, a subject treated for ASMD in accordance with the methodsprovided herein is a human that has had a major organ transplant (e.g.,liver or bone marrow transplant). In some embodiments, a subject treatedfor ASMD in accordance with the methods provided herein is a human thathas not had a total or partial splenectomy. In other embodiments, asubject treated for ASMD in accordance with the methods provided hereinis a human that has had a total or partial splenectomy.

In certain embodiments, a subject treated for ASMD in accordance withthe methods provided herein is a human that does not have or has notbeen diagnosed with one or more of the following: active hepatitis B,active hepatitis C, human immunodeficiency virus (HIV) infection,cirrhosis, or significant cardiac disease (e.g., moderate or severepulmonary hypertension or valvular dysfunction, or less than 50%, lessthan 40%, less than 30% or less than 20% left ventricular ejectionfraction by echocardiography). In other embodiments, a subject treatedfor ASMD in accordance with the methods provided herein is a human thathas or has been diagnosed as having one or more of the following: activehepatitis B, active hepatitis C, an human immunodeficiency virus (HIV)infection, cirrhosis, or significant cardiac disease (e.g., moderate orsevere pulmonary hypertension or valvular dysfunction, or less than 50%,less than 40%, less than 30% or less than 20% left ventricular ejectionfraction by echocardiography).

In certain embodiments, a subject treated for ASMD in accordance withthe methods provided herein is a human that does not have one or more ofthe following: an International Normalized Ratio (INR) of greater than1.25, 1.5, 1.75 or 2, a platelet count of less than 60×10³ per analanine aminotransferase (ALT) of greater than 250 IU/L, an aspartateaminotransferase of greater than 250 IU/L, or a total bilirubin greaterthan 1.5 mg/dL, 1.75 mg/dL or 2 mg/dL. In other embodiments, a subjecttreated for ASMD in accordance with the methods provided herein is ahuman that has one or more of the following: an International NormalizedRation (INR) of greater than 1.25, 1.5, 1.75 or 2, a platelet count ofless than 60×10³ per an alanine aminotransferase (ALT) of greater than250 IU/L, an aspartate aminotransferase of greater than 250 IU/L, or atotal bilirubin greater than 1.5 mg/dL, 1.75 mg/dL or 2 mg/dL.

In certain embodiments, a subject treated for ASMD in accordance withthe methods provided herein is a human that is not taking one or more ofthe following medications: chlorpromazine, imipramine or desipramine. Inother embodiments, a subject treated for ASMD in accordance with themethods provided herein is a human that is taking one or more of thefollowing medications: chlorpromazine, imipramine or desipramine.

In certain embodiments, a subject treated for ASMD in accordance withthe methods provided herein is a human that is not taking herbalsupplements or medications that may cause or prolong bleeding (e.g.,anti-coagulants, ibuprofen, aspirin, garlic supplements, ginkgo, andginseng), or have potential hepatotoxicity (e.g., 3-hydroxy-3-methylglutaryl [HMG]-CoA reductase inhibitors, erythromycin, valproic acid,anti-depressants, kava and echinaecea). In other embodiments, a subjecttreated for ASMD in accordance with the methods provided herein is ahuman that is taking one or more of the following herbal supplementalsor medications: anti-coagulants, ibuprofen, aspirin, garlic supplements,ginkgo, ginseng, 3-hydroxy-3-methyl glutaryl [HMG]-CoA reductaseinhibitors, erythromycin, valproic acid, anti-depressants, kava andechinaecea.

In specific embodiments, a subject treated for ASMD in accordance withthe methods provided herein is human that meets one, two or more, or allof the criteria for subjects in the working examples in Sections 6, 8,and 9 et seq.

5.3. Monitoring Treatment

In accordance with the method provided herein, a number of parameters(e.g., factors or markers) can be monitored before, during and/or afterthe administration of a dose of ASM. In certain embodiments, a physicalexamination is performed prior to administration of ASM and as needed orrecommended during the course of treatment with ASM. A physicalexamination can include the following assessments: general appearance,skin, head, ears, eyes, nose, and throat (HEENT), lymph nodes, heart,lungs, abdomen, extremities/joints, neurological, mental status, andreflexes. In certain embodiments, vital signs, continuous heart rate,respiratory rate, temperature and oxygen saturation can be assessedbefore, during and/or after the administration of a dose of ASM. Heartrates can be monitored continuously by telemetry beginning, e.g., 6hours before administration of a dose of ASM up to 72 hours afteradministration of a dose of ASM.

In specific embodiments, a complete blood count with differential, bloodurea nitrogen (BUN), bicarbonate, creatinine, glucose, uric acid,calcium, phosphate, albumin, total protein, sodium, potassium, chloride,lactate dehydrogenase, creatine kinase, creatine kinase with MBfraction, and urinalysis (including urine color, appearance, specificgravity, pH, protein, glucose, ketones, bilirubin, hemoglobin, andmicroscopy if indicated) can be performed at the times specified before,during and/or after the administration of a dose of ASM. In certainembodiments, liver tests, coagulation studies, and/or fasting lipidprofile can be performed before, during and/or after the administrationof a dose of ASM. Liver functions tests can include assessingconcentrations of alanine aminotransferase (ALT), aspartateaminotransferase (AST), alkaline phosphatase (AP), gamma-glutamyltransferase (GGT) and total and direct bilirubin. Coagulation studiescan include assessing prothrombin time (PT), partial thromboplastin time(PTT), International normalized ratio (INR), D-dimer concentration, andfibrinogen concentration. Fasting lipid profile assessments can includeassessments of total cholesterol (TC), low-density lipoprotein (LDL),high-density lipoprotein (HDL), very low-density lipoprotein (VLDL) andtriglycerides.

In certain embodiments, a skin biopsy is performed prior to, duringand/or subsequent to the administration of a dose of ASM. In someembodiments, a liver biopsy is performed prior to, during and/orsubsequent to the administration of a dose of ASM. Sphingomyelin levelsin skin and liver biospsies can be assessed by metamorph histologicalanalysis.

In certain embodiments, pulmonary function tests are performed prior to,during and/or after the administration of a dose of ASM. Pulmonaryfunction testing equipment calibration and test administration protocolscan be standardized accordance with American Thoracic Society (ATS)guidelines (ATS, 1991, Am Rev Respir Dis 144: 1202-1218). In certainembodiments, percent predicted forced vital capacity (FVC) is assessedprior to, during and after the administration of a dose of ASM. FVC isthe total volume of air expired during a forced maneuver. The FVC can bemeasured using standard spirometric techniques.

In certain embodiments, forced expiratory volume in 1 second (FEV1) canbe performed prior to, during and after the administration of a dose ofASM. FEV1 is the volume of air expelled during the first second of FVC.The FEV1 can be measured using standard spirometric techniques.

In certain embodiments, total lung capacity (TLC) can be assessed priorto, during and after the administration of a dose of ASM. TLC is thetotal volume of air within the lungs following a maximal inspiratoryeffort. The TLC can be measured using whole body plethysmography.

In certain embodiments, DLco can be assessed prior to, during and afterthe administration of a dose of ASM. DLco measures the rate of diffusionof a diffusion-limited gas (carbon monoxide, CO) per minute across thealveolocapillary membrane. DLco can be calculated by comparing theamount of carbon monoxide (CO) exhaled following a known amount ofinhaled CO. Helium, which does not diffuse across the alveolocapillarymembrane, can be included as a tracer with the inspired CO to controlfor air trapping.

In certain embodiments, a chest X-ray (posterior-anterior and lateral)can be obtained prior to, during and/or after the administration of adose of ASM. In certain embodiments, an abdominal Mill is obtainedbefore, during and/or after the administration of a dose of ASM.

In certain embodiments, a blood sample is collected before, duringand/or after administration of a dose of ASM for assessment ofbiomarkers, bilirubin concentration, and the percentage of neutrophilsof total white blood cells. In a specific embodiment, the concentrationof one or more of the following biomarkers is assessed using techniquesknown to one skilled in the art: CRP/hs-CRP concentration, sphingomyelinconcentration, iron concentration, ferritin concentration, calcitoninconcentration, albumin concentration, SAA, serum amyloid P component,ACE, CCL18, chitotriosidase, transferrin, fibrinogen concentration, andplasma sphingomyelin concentration, plasma ceramide concentration. Incertain embodiments, the concentration of one or more of the followingcytokines is assessed using techniques known to one skilled in the art:IL-6 and IL-8.

In certain embodiments, the concentration of antibodies against ASM(e.g., anti-recombinant human ASM IgG and/or anti-recombinant human ASMIgE) is assessed before, during and/or after the administration of adose of ASM. In some embodiments, the concentration of one, two or morecomplement factors are assessed before, during and/or after theadministration of a dose of ASM. In certain embodiments, theconcentration of serum tryptase is assessed before, during and/or afterthe administration of a dose of ASM. In certain embodiments, a skin testto determine IgE-mediated reactions to ASM is assessed before, duringand/or after the administration of a dose of ASM.s.

In certain embodiments, the pharmacokinetic profile for ASM is measuredbefore, during and/or after the administration of a dose of ASM. In someembodiments, the BAL cell count is measured before, during and/or afterthe administration of a dose of ASM. See Section 8 et seq, infra, forother parameters that may be assessed before, during and/or after theadministration of a dose of ASM.

In certain embodiments, a factor or marker described herein is assessed1 week, 72 hours, 48 hours, 24 hours, 12 hours, 6 hours, 4 hours, 2hours, 1 hour, 30 minutes or 15 minutes before the administration of adose of ASM. In some embodiments, a factor or marker described herein isassessed during the administration of a dose of ASM. For example, afactor or marker described herein is assessed during the administrationof a dose of ASM. In certain embodiments, a factor or marker describedherein is assessed 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6hours, 8 hours 12 hours, 16 hours, 18 hours, 24 hours, 36 hours, 48hours, 72 hours, 4 day, 5 days, 6 days, 1 week, two weeks, three weeks,four weeks, one month, two months, three months or longer after theadministration of a dose of ASM. In certain embodiments, a factor ormarker described herein is assessed some many hours or weeks after acertain number of doses of ASM. For example, a factor or markerdescribed herein may be assessed every four weeks, month, 2 months, 3months, 4 months, 5 months or 6 months after a certain number of dosesof ASM. In certain embodiments, a factor or marker is assessed every 4weeks, month, 2 months, 3 months, 4 months 5 months, 6 months, 7 months,8 months, 9 months, 10 months, 11 months, or 12 months. In someembodiments, a factor or marker is assessed every month to 2 months,every month to 4 months, every 2 months to 4 months, every 2 months to 4months, every 3 months to 4 months, every 2 months to 5 months, every 3months to 5 months, every 4 months to 5 months, every 2 months to 6months, every 3 months to 6 months, every 4 months to 6 months, or every5 months to 6 months. In certain embodiments, a factor or marker isassessed every 6 months to 8 months, every 6 months to 12 months, every8 months to 12 months, every 9 months to 12 months, or every 10 monthsto 12 months. In some embodiments, one or more factors or markersdescribed herein is not assessed before, during and/or after theadministration of a dose of ASM.

In certain embodiments, the results from the assessment of one or morefactors or markers indicates that the dosage of ASM should be adjusted.

5.4. Biological Samples

In accordance with the methods described herein, a biological sample issubjected to one or more pretreatment steps prior to the detectionand/or measurement of a cell population, factor or marker (e.g.,biomarker). In certain embodiments, a biological fluid is pretreated bycentrifugation, filtration, precipitation, dialysis, or chromatography,or by a combination of such pretreatment steps. In other embodiments, atissue sample is pretreated by freezing, chemical fixation, paraffinembedding, dehydration, permabilization, or homogenization followed bycentrifugation, filtration, precipitation, dialysis, or chromatography,or by a combination of such pretreatment steps. In certain embodiments,the sample is pretreated by removing cells of a certain type from thesample, or removing debris from the sample prior to the determination ofthe number or amount of a particular cell type(s) in the sampleaccording to the methods described herein.

The biological sample can be a tissue sample, biological fluid,discharge, or any other sample from a human subject. In someembodiments, the biological sample is a blood sample or bone marrowsample. In certain embodiments, the biological sample is a tissue sample(e.g., a liver, skin or lung biopsy). In some embodiments, thebiological sample is a biological fluid such as urine. In certainembodiments, the biological sample is a sputum or nasal dischargesample. In some embodiments, the biological sample is mouth swab.

Techniques known in the art can be used to assess the presence, number,amount or percentage of a certain type(s) of cells present in abiological sample. For example, cells can be sorted using a using afluorescence activated cell sorter (FACS). Fluorescence activated cellsorting (FACS) is a known method for separating particles, includingcells, based on the fluorescent properties of the particles. See, forexample, Kamarch, 1987, Methods Enzymol 151:150-165. Laser excitation offluorescent moieties in the individual particles results in a smallelectrical charge allowing electromagnetic separation of positive andnegative particles from a mixture. An antibody or ligand used to detectan antigenic determinant present on the cell surface of particular cellsis labeled with a fluorochrome, such as FITC or phycoerythrin. The cellsare incubated with the fluorescently labeled antibody or ligand for atime period sufficient to allow the labeled antibody or ligand to bindto cells. The cells are processed through the cell sorter, allowingseparation of the cells of interest from other cells. FACS sortedparticles can be directly deposited into individual wells of microtiterplates to facilitate separation.

Magnetic beads can be also used to separate cells. For example, cellscan be sorted using a using a magnetic activated cell sorting (MACS)technique, a method for separating particles based on their ability tobind magnetic beads (0.5-100 μm diameter). A variety of usefulmodifications can be performed on the magnetic microspheres, includingcovalent addition of an antibody which specifically recognizes acell-solid phase surface molecule or hapten. A magnetic field is thenapplied, to physically manipulate the selected beads. In a specificembodiment, antibodies to a blood cell surface marker are coupled tomagnetic beads. The beads are then mixed with the blood cell culture toallow binding. Cells are then passed through a magnetic field toseparate out cells having the blood cell surface markers of interest.These cells can then be isolated.

In some embodiments, the surface of a culture dish may be coated withantibodies, and used to separate cells by a method called panning.Separate dishes can be coated with antibody specific to particularcells. Cells can be added first to a dish coated with blood cellspecific antibodies of interest. After thorough rinsing, the cells leftbound to the dish will be cells that express the cell markers ofinterest. Examples of cell surface antigenic determinants or markersinclude, but are not limited to, CD2 for T lymphocytes and naturalkiller cells, CD3 for T lymphocytes, CD11a for leukocytes, CD28 for Tlymphocytes, CD19 for B lymphocytes, CD20 for B lymphocytes, CD21 for Blymphocytes, CD22 for B lymphocytes, CD23 for B lymphocytes, CD29 forleukocytes, CD14 for monocytes, CD41 for platelets, CD61 for platelets,CD66 for granulocytes, CD67 for granulocytes and CD68 for monocytes andmacrophages.

The presence, concentration or amount of a marker (including abiomarker) or factor can be assessed used techniques known in the art.The presence, concentration or amount of a marker (including abiomarker) or factor can be measured at the protein level and/or RNAlevel using techniques known to one skilled in the art. At the proteinlevel, immunoassays, such as ELISAs and immunopreciptation and westernblots can be used to measure the presence, concentration or amount of amarker (including a biomarker) or factor. In addition, FACS can be usedto measure the presence, concentration or amount of a marker (includinga biomarker) or factor. At the RNA level, RT-PCR and Northern blots canbe used to measure the presence, concentration or amount of a marker(including a biomarker) or factor.

5.5. Co-Therapies

In some embodiments, a method for treating ASMD involves theadministration of ASM in combination with one or more additionaltherapies. As used herein, the term “in combination,” refers, in thecontext of the administration of ASM, to the administration of a doseASM prior to, concurrently with, or subsequent to the administration ofone or more additional therapies (e.g., agents) for use in treatingASMD. The use of the term “in combination” does not restrict the orderin which ASM and one or more additional therapies are administered to asubject. In specific embodiments, the interval of time between theadministration of a dose of ASM and the administration of one or moreadditional therapies may be about 1-5 minutes, 1-30 minutes, 30 minutesto 60 minutes, 1 hour, 1-2 hours, 2-6 hours, 2-12 hours, 12-24 hours,1-2 days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10weeks, 15 weeks, 20 weeks, 26 weeks, 52 weeks, 11-15 weeks, 15-20 weeks,20-30 weeks, 30-40 weeks, 40-50 weeks, 1 month, 2 months, 3 months, 4months 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11months, 12 months, 1 year, 2 years, or any period of time in between. Incertain embodiments, a dose ASM and one or more additional therapies areadministered less than 1 day, 1 week, 2 weeks, 3 weeks, 4 weeks, onemonth, 2 months, 3 months, 6 months, 1 year, 2 years, or 5 years apart.

In some embodiments, the combination therapies provided herein involveadministering a dose of ASM every 2 to 4 weeks, and administering one ormore additional therapies once a week, once every 2 weeks, once every 3weeks, once every 4 weeks, once every month, once every 2 months (e.g.,approximately 8 weeks), once every 3 months (e.g., approximately 12weeks), or once every 4 months (e.g., approximately 16 weeks). Incertain embodiments, ASM and one or more additional therapies arecyclically administered to a subject. Cycling therapy involves theadministration of ASM for a period of time, followed by theadministration of one or more additional therapies for a period of time,and repeating this sequential administration. In certain embodiments,cycling therapy may also include a period of rest where ASM or theadditional therapy is not administered for a period of time (e.g., 2days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks,4 weeks, 5 weeks, 10 weeks, 20 weeks, 1 month, 2 months, 3 months, 4months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11months, 12 months, 2 years, or 3 years). In an embodiment, the number ofcycles administered is from 1 to 12 cycles, from 2 to 10 cycles, or from2 to 8 cycles.

In some embodiments, the methods for treating ASMD provided hereincomprise administering ASM as a single agent for a period of time priorto administering ASM in combination with an additional therapy. Incertain embodiments, the methods for treating ASMD provided hereincomprise administering an additional therapy alone for a period of timeprior to administering ASM in combination with the additional therapy.

In some embodiments, the administration of ASM and one or moreadditional therapies in accordance with the methods presented hereinhave an additive effect relative the administration of ASM or said oneor more additional therapies alone. In some embodiments, theadministration of ASM and one or more additional therapies in accordancewith the methods presented herein have a synergistic effect relative tothe administration of ASM or said one or more additional therapiesalone.

As used herein, the term “synergistic,” refers to the effect of theadministration of a dose ASM in combination with one or more additionaltherapies (e.g., agents), which combination is more effective than theadditive effects of any two or more single therapies (e.g., agents). Ina specific embodiment, a synergistic effect of a combination therapypermits the use of lower dosages (e.g., sub-optimal doses) of ASM or anadditional therapy and/or less frequent administration of ASM or anadditional therapy to a subject. In certain embodiments, the ability toutilize lower dosages of ASM or of an additional therapy and/or toadminister ASM or said additional therapy less frequently reduces thetoxicity associated with the administration of ASM or of said additionaltherapy, respectively, to a subject without reducing the efficacy of ASMor of said additional therapy, respectively, in the treatment of ASMD.In some embodiments, a synergistic effect results in improved efficacyof ASM and each of said additional therapies in treating ASMD. In someembodiments, a synergistic effect of a combination of ASM and one ormore additional therapies avoids or reduces adverse or unwanted sideeffects associated with the use of any single therapy.

In particular embodiments, one or more additional therapies areadministered in combination with ASM to subjects to reduce or ameliorateone or more of the following that may be associated with theadministration of a particular dose of ASM: (i) a related adverse event,(ii) a total bilirubin value greater than the bilirubin value for ahuman without ASMD (e.g., a healthy human), (iii) a plasma ceramideconcentration greater than the plasma ceramide concentration of a humanwithout ASMD (e.g., a healthy human), or (iv) an acute phase response.In specific embodiments, one or more additional therapies areadministered in combination with ASM to subjects to increase pulmonaryfunction while minimizing one or more of the following that may beassociated with the administration of a particular dose of ASM: (i) arelated adverse event, (ii) a total bilirubin value greater than thebilirubin value for a human without ASMD (e.g., a healthy human), (iii)a plasma ceramide concentration greater than the plasma ceramideconcentration of a human without ASMD (e.g., a healthy human), or (iv)an acute phase response.

In certain embodiments, one or more additional therapies administered incombination with ASM to a subject to control or relieve symptomsassociated with ASMD. In some embodiments, one or more additionaltherapies that are administered in combination with ASM to a subject arepain relievers. Specific examples of therapies can be administered incombination with ASM include, but are not limited to,N-Acetyl-L-cysteine (NAC), S-Adenosyl-L-methionine (SAM), interleukin(IL)-6 antibody, IL-6 receptor antibody, dexamethasone, L-Nil, (aselective inhibitor of inducible NOS), L-NAME (a selective inhibitor ofNOS), basic fibroblast growth factor (b-FGF), imipramine (asphingomyelinase inhibitor), D609 (a sphingomyelinase inhibitor), andN-oleoylethanolamine (NOE; a ceramide inhibitor).

In certain embodiments, chaperones, such as small molecule chaperones,are administered in combination with ASM. See, e.g., U.S. Pat. No.7,750,050 and International Publication Nos. WO 2004/045574 and WO2010/015816, which are incorporated by reference herein, for agents(e.g., small molecules) that may be administered in combination with ASMto a subject. In some embodiments, the chaperone (e.g., small moleculechaperone) does one, two or all of the following: increases thetargeting of ASM to sites of pathology, stabilizes the activity of theASM, and enhances the activity of the ASM.

In some embodiments, a glucocorticosteroid, such as dexamethasone, areadministered in combination with ASM. See, e.g., U.S. Pat. No.7,658,916, which is incorporated herein by reference in its entirety,for agents (e.g., glucocorticosteriods) that can be administered incombination with ASM.

In some embodiments, a substrate reduction molecule is administered incombination with ASM to a subject. In specific embodiments, a molecule(e.g., a small molecule) that either decreases the amount ofsphingomyelin, reduces the rate of sphingomyelin synthesis, or both isadministered in combination with ASM to a subject. See, e.g., Li et al.2007, Biochim Biophys Act 1771(9):1186-1194 (which is incorporatedherein by reference in its entirety) for sphingomyelin synthaseinhibitors, such as tricyclodecan-9-yl-xanthogenate and sphingomyelinsynthase siRNAs.

In some embodiments, a therapy that reduces the potential immunogenicityof ASM is administered in combination with ASM to a subject. In certainembodiments, benedryl is administered in combination with ASM to asubject.

The combination of ASM and one or more additional therapies can beadministered to a subject in the same pharmaceutical composition.Alternatively, ASM and one or more additional therapies can beadministered concurrently to a subject in separate pharmaceuticalcompositions. ASM and one or more additional therapies can beadministered sequentially to a subject in separate pharmaceuticalcompositions. ASM and one or more additional therapies may also beadministered to a subject by the same or different routes ofadministration.

5.6. Pharmaceutical Products

In one aspect, described herein are finished packaged and labeledpharmaceutical products. In one embodiment, a pharmaceutical productcomprises a unit dosage form of ASM in an appropriate vessel orcontainer (e.g., a glass vial or other container that is hermeticallysealed). In some embodiments, the unit dosage form is a lyophilized formof ASM, and under those circumstances, the pharmaceutical product maycontain a second container with sterile saline or sterile water forreconstituting the lyophilized form of ASM. In other embodiments, theunit dosage form is an aqueous form of ASM that does not requirereconstitution before administration to a subject. In specificembodiments, the unit dosage form of ASM contains ASM in an amountsufficient for the administration of a low, non-toxic dose of the enzymeto a subject. In certain embodiments, the unit dosage form is suitablefor the selected route of administration of the ASM to the subject. In aspecific embodiment, the unit dosage form is suitable for intravenousdelivery to a subject.

In one embodiment, a pharmaceutical product comprises a unit dosage formof ASM in an appropriate vessel or container (e.g., a glass vial orother container that is hermetically sealed) and an infusion pump orsyringe pump for the administration of the ASM to a subject.

As with any pharmaceutical product, the packaging material and containerare designed to protect the stability of the product during storage andshipment. Further, the pharmaceutical product includes instructions foruse or other informational material that advise the physician,technician or patient on how to appropriately treat ASMD. In otherwords, the pharmaceutical product includes instruction means indicatingor suggesting a dosing regimen including, but not limited to, actualdoses (e.g., a dose escalation protocol), monitoring procedures, andother monitoring information. In certain embodiments, the pharmaceuticalproduce includes a means for genotyping a patient (e.g., PCR primers forthe SMPD1 gene).

6. EXAMPLE 1: HUMAN TRIAL OF RECOMBINANT HUMAN ACID SPHINGOMYELINASE(RHASM) ENZYME REPLACEMENT THERAPY IN HUMAN ADULTS WITH ASM DEFICIENCY6.1. Introduction

ASMD is an autosomal recessive, lysosomal storage disorder that resultswhen sphingomyelin is unable to be normally catabolized to ceramide andphosphorylcholine. Consequently, sphingomyelin accumulates within cellsprimarily of the reticuloendothelial system, leading tohepatosplenomegaly, anemia, thrombocytopenia, and interstitial lungdisease. Growth retardation and an atherogenic lipid profile also arecommon findings. Patients who have little to no residual ASM activityexhibit the most severe symptoms with onset in infancy, failure tothrive, neurodegeneration, and death by age 3 (Niemann-Pick disease typeA, NPD A). Patients with higher amounts of residual ASM activity havevariable ages of onset, heterogeneous presentations and somaticsymptoms, little to no neurologic involvement, and generally surviveinto adulthood (NPD B). Currently, there is no treatment for patientswith ASMD.

Enzyme replacement therapy (ERT) has been successfully used to treatseveral lysosomal storage disorders, including Gaucher disease,Mucopolysaccharidosis types I, II, and VI, Fabry disease, and Pompedisease. Recombinant human lysosomal enzymes are administeredintravenously and taken up into cells by receptor-mediated endocytosisfor subsequent targeting to lysosomes. Proof of principle for thetreatment of ASMD was demonstrated by Dr. Edward Schuchman's laboratory(Mount Sinai Medical Center) in an ASM knockout mouse (ASMKO) modelwhere intravenous injections of recombinant human ASM (rhASM)efficiently reduced sphingomyelin levels in liver and spleen, and to alesser extent in lung (Miranda, et al., FASEB 2000; 14:1988). However,sphingomyelin levels were not reduced in brain because of the inabilityof rhASM to cross the blood-brain barrier. Due to the fact that theASMKO mouse has no residual ASM activity or protein and develops rapidand severe neurological disease, this animal is considered mostappropriately a model of NPD—type A (see, Buccinna et al., 2009, J.Neurochem. 109:105-115).

Additional studies confirmed that biweekly doses of rhASM reducedsphingomyelin levels in ASMKO mice in a dose-dependent manner (0.3-3mg/kg). The no observed adverse effect levels (NOAEL) for single andrepeat dosing were determined to be 0.3 and 3 mg/kg, respectively inASMKO mice. Subsequent attempts to deplete sphingomyelin levels in lungwith higher doses of rhASM led to unexpected toxicity. At doses ≥10mg/kg, ASMKO mice but not normal animals experienced liver inflammation,adrenal hemorrhage, cardiovascular shock and death in the setting ofvery elevated cytokine levels, suggesting cytokine release syndrome. Thetoxicity and cytokine elevations seen with high doses of rhASM could beameliorated or prevented by prior treatment of ASMKO mice with severallower doses of rhASM, suggesting that the rate and amount ofsphingomyelin degradation plays a key role.

In the ASMKO mouse, a model for NPD-type A, toxicity was not observedfollowing single doses of ≤0.3 mg/kg rhASM and repeat doses of ≤ of 3.0mg/kg rhASM; severe toxicity was not observed until single doses ≥10mg/kg rhASM were administered. Therefore, a conservative starting doseof 0.03 mg/kg rhASM was selected for single-dose treatment of humansubjects to ensure a 10-fold safety margin with respect to thesingle-dose NOAEL (0.3 mg/kg) observed in ASMKO mice. A maximum dose of1.0 mg/kg rhASM was selected for single dose treatment of human subjectsto ensure a 10-fold safety margin with respect to the dose at whichserious toxicity was observed in the ASMKO mouse (10 mg/kg). Uponcompletion of the protocol set forth below, toxicity in the humansubjects was unexpectedly observed with a dose as low as 0.3 mg/kgrhASM.

6.2. Materials and Methods 6.2.1. Human Protocol Design

This protocol was a single-center, single-dose, dose escalation Phase 1trial. The primary trial objectives were to evaluate the safety andpharmacokinetics of single doses of rhASM in human adults withnon-neuronopathic ASMD (Niemann-Pick B). Single doses of 0.03, 0.1, 0.3,0.6 and 1.0 mg/kg rhASM were infused sequentially by dose cohort. Theoriginal trial design called for a minimum of 15 patients (5 cohorts of3 patients each). Due to difficulties with patient enrollment, theprotocol was amended such that the first 2 cohorts enrolled 3 patientseach and the last 3 cohorts were to enroll 2 patients each. Anindependent data monitoring committee oversaw the conduct of the trialand all protocol procedures were approved by the IRB. FIG. 1 depicts theprotocol patient flow.

6.2.2. Patients

To be eligible for the protocol, patients had to be 18-65 years of ageand have deficient ASM enzyme activity, a spleen volume ≥2× normal, ASTand ALT ≤250 IU/L, bilirubin ≤3.6 mg/dL, INR ≤1.5, DLco>30% predicted,and platelets ≥60,000/mL. Patients were excluded if they had cirrhosis(by liver biopsy), significant cardiac disease, total splenectomy, orwere taking medications or herbal supplements that were potentiallyhepatotoxic, promoted bleeding, or inhibited rhASM.

A total of 13 patients were enrolled and 11 patients infused with rhASM.rhASM was produced by over-expression of ASM cDNA in Chinese hamsterovary cells. The mean age of infused patients was 30.8 yrs, all wereCaucasian (non-Hispanic/non-Latino), and mean spleen volume was 10.8multiples of normal. One had a partial splenectomy; the remainingpatients had intact spleens at protocol entry. FIG. 2 depicts thedemography and baseline characteristics of the patients in thisprotocol.

Once screening was completed and eligibility was confirmed, patientswere admitted to the cardiac care unit (CCU) overnight for baselinetelemetry and infused the following morning with rhASM. Patients weremonitored for 72 hrs post-dose while on telemetry (24 hrs in the CCU and48 hrs in the General Clinical Research Center). Patients returned foran overnight visit on Day 14 and an outpatient visit on Day 28.

The following sets forth the medical assessments that were made duringthe protocol:

-   -   Physical exam—Days 0, 1, 2, 14, and 28    -   Chemistries, hematology, and urinalysis—preinfusion, then 24,        48, 72 hrs; Days 14, 28    -   Liver function tests—preinfusion, then q12 hrs through 72 hrs,        Days 14, 28, Aldosterone, cortisol,        delta-4-androstenedione-preinfusion, then q12 hrs through 72 hrs    -   ACTH stimulation test—Screening, Day 14 Telemetry—continuous        through 72 hrs ECG, echocardiogram-preinfusion, end of infusion,        and postinfusion at 1, 2, 6, 12, and 4 hrs, Day 14    -   Cardiac biomarkers (BNP, cardiac troponin-I,        CPK-MB)-preinfusion, then 2, 6, 12, 24 hrs    -   Sphingomyelin, ceramide levels in plasma—preinfusion, then 24,        48, 72 hrs; Days 14 and 28    -   Pulmonary function testing and CXR—Screening, Day 14    -   Liver and spleen volumes by MM—Screening, Day 14    -   Liver and skin biopsies—Screening, Day 14    -   Biomarkers—predose, Days 14, 28    -   Anti-rhASM IgG testing—preinfusion, Day 28    -   Pharmacokinetics—within 30 min preinfusion, 15 min after start        of infusion, end of infusion, and at following time points        post-infusion: 15 min, 30 min, 45 min; 1 hr., 2, 3, 4, 6, 8, 12,        18, 24, 48, 72 hrs.    -   Cytokines (IL-1a, IL-1b, IL-6, G-CSF, GM-CSF, MIP-1a,        TNF-a)—pharmacokinetic time points (above) plus Day 14

6.3. Results

As depicted in FIGS. 3A and 3B, plasma ceramide levels and plasmasphingomyelin levels, respectively, were determined at several timepoints. Plasma ceramide levels showed a dose-dependent rise by 6 hrs andpeaked at 18-72 hrs. Plasma sphingomyelin levels were normal at baselineand showed no consistent trend over time. In patient no. 12112 whoreceived the highest dose (1 mg/kg), the plasma sphingomyelin level rosepost-dose and peaked at 72 hours.

FIG. 4 depicts total bilirubin levels determined over several timepoints during the protocol. Total bilirubin showed a dose-related riseby 24 hrs and peaked at 48-60 hrs. The highest total bilirubin was 4.7mg/dL in patient no. 12112 who received the highest dose (1 mg/kg).There were proportional increases in direct and indirect bilirubin. Noincreases were seen in ALT, AST, or alkaline phosphatase. There was amild increase in GGT through 72 hrs (not shown) in two patients whoreceived the highest (1 mg/kg, patient no. 12112) and lowest (0.03mg/kg, patient no. 10503) doses. Hemoglobin and hematocrit levels werestable through the protocol, indicating that hemolysis was notresponsible for elevated bilirubin levels.

FIGS. 5A-5G depict the levels of acute phase response (inflammation)markers, CRP/hs-CRP, % neutrophils, fibrinogen, ferritin, IL-8, IL-6,and calcitonin, respectively, determined at several time points forseveral patients. CRP/hs-CRP showed a transient dose-related rise by 24hrs, peaked at 48-72 hrs, and returned to normal by Day 14. Other acutephase reactants also showed increases (% neutrophils, fibrinogen,ferritin, PT and PTT) and decreases (iron, albumin). Certaininflammatory biomarkers, e.g., the cytokines IL-6 and IL-8, andcalcitonin, also showed substantial elevations that were dose dependentand peaked 48 hours post-infusion. Among these inflammatory mediators,the greatest changes in descending order occurred in IL 8 (peak 33.8×upper limit of normal), calcitonin (peak 33.4× upper limit of normal),CRP (peak 9.8× upper limit of normal), and ferritin (3.9× upper limit ofnormal). There was no trend in platelet count or the level offibrin-split products (not shown). The rises in laboratory acute phasereactants correlated with the constitutional clinical symptoms of fever,nausea, vomiting, headache, pain, and myalgias in some patients.

FIG. 6 is a chart of treatment emergent adverse events for four patientseach on a different dose of rhASM, which events were considered to berelated (possibly, probably, or definitely) to treatment. As set forthin the chart, patient no. 11509, who was given a dose of 0.3 mg/kgrhASM, exhibited moderately severe adverse events starting on day 2.

With regard to related adverse events reported in the protocol, therewere no significant cardiovascular changes by telemetry, ECG,echocardiogram, or biomarkers (BNP, cardiac troponin-I, CPK-MB) oradrenal hormone dysfunction. One patient (no. 12112, 1.0 mg/kg cohort)had an elevated cortisol level in the morning 24 hours post-dose, whichmost likely represented a normal physiologic stress response to severalongoing moderate related adverse events. Further, four of six patientsreceiving ≥0.3 mg/kg rhASM experienced a total of 19 clinical andlaboratory related adverse events assessed as drug-related. Theintensity of related adverse events ranged from mild to severe, but mostwere moderate and did not require any intervention. They included fever(n=2), pain [myalgia; abdominal, leg, and hip pain] (n=2), nausea (n=2),scleral icterus and urine urobilinogen (n=1), fatigue (n=1), vomiting(n=1), lymphocytic infiltrate/hepatocellular degeneration on liverbiopsy (n=1), acute phase reaction (n=4), elevated bilirubin (n=2), andincreased fibrin D-dimer (n=1). The onset of clinical symptoms began 12hours post-infusion and resolved by 72 hrs, except for hip pain in onepatient that began after 72 hours. At day 14, a liver biopsy in onepatient (0.6 mg/kg rhASM, patient no. 12313) showed two new foci oflymphocytic infiltrates: one was tiny (0.1 mm diameter), and the otherwas moderate (0.5 mm diameter) and which was associated withhepatocellular degeneration. This protocol was stopped when the firstpatient in cohort 5 (1 mg/kg rhASM, patient no. 12112) experienced adose-limiting toxicity of hyperbilirubinemia (peak 4.7 mg/dL).

The results of this showed an unexpected and delayed onset ofdose-related clinical and laboratory adverse events in humans sufferingfrom ASMD at doses of rhASM of 0.3 mg/kg and higher. Further, two majorsafety laboratory observations were made in view of the results of thisprotocol. One regarding hyperbilirubinemia, in which a proportionate todose rise in direct and indirect bilirubin was observed with noconsistent markers of liver damage (AST, ALT, AP) or evidence ofhemolysis (hemoglobin and hematocrit). Two patients had mildly elevatedGGT at 48-72 hours, and one patient had two new liver foci oflymphocytic infiltrates, one of which was associated with hepatocellulardegeneration.

The other observation concerned the acute phase response, i.e.,inflammation. The results showed an increase in CRP/hs-CRP, %neutrophils, ferritin, IL-6, IL-8, calcitonion, fibrinogen, PT, PTT, anda decrease in iron and albumin in response to increasing dosages ofrhASM. The constitutional clinical symptoms of fever, nausea, vomiting,pain, and myalgia were most likely related to the acute phase response.The results showed no evidence of cytokine release syndrome associatedwith cardiovascular changes, and no evidence of adrenal hormonedysfunction.

6.4. Conclusions

The above-described protocol is the first experience with enzymereplacement therapy in adult human patients with ASMD. At bioactivedoses in humans, rhASM did not cause cytokine release syndromeassociated with cardiovascular changes, or cause adrenal hormonedysfunction. The major safety observations were dose-relatedhyperbilirubinemia and acute phase response. Both adverse events arelikely related to the breakdown of sphingomyelin into ceramide andphosphylcholine, but the exact molecular mechanisms are not fullyunderstood. Several safety biomarkers were identified, includingbilirubin, ceramide, hsCRP, IL-8, IL-6, calcitonin, and ferritin.Further, based on the hyperbilirubinemia findings, the maximum toleratedstarting dose of rhASM was 0.6 mg/kg.

Moreover, the fact that the onset of adverse events with clinicalsymptoms in patients was observed at a dose as low as 0.3 mg/kg wassurprising, given that the NOAEL (“no observed adverse effect level”) inthe ASMKO mouse was 0.3 mg/kg. Notably, clinical symptoms of toxicity inthe ASMKO mice was not observed until doses greater than or equal to 10mg/kg were used.

7. EXAMPLE 2: REPEAT DOSE INTRAVENOUS INJECTION TOXICITY PROTOCOLFOLLOWING DEBULKING PHASE IN ACID SPHINGOMYELINEASE KNOCK-OUT MICE

This example describes the investigation of the potential toxicity ofrepeated intravenous administration of recombinant human acidsphingomyelinase (rhASM) following a debulking phase in acidsphingomyelinase knockout (ASMKO) mice. Having established that toxicside effects begin to be observed at intital doses of 10 mg/kg of rhASMin ASMKO mice, the following investigation was designed to determine ifthe administration of escalating doses of recombinant human acidsphingomyelinase (rhASM) in ASMKO mice would debulk enough of theaccumulated sphingomyelin so that a higher dose of rhASM could beadministered to the ASMKO mice to target sites of pathology, such as thelung and brain, with minimal or no observable toxicity.

JK ASMKO mice were administered 3 mg/kg rhASM on study days (SD) 1, 3,5, 7 (Debulking Phase). Beginning on SD 9 and continuing every otherweek for 13 weeks (7 doses) mice received treatment doses of 3, 10, or30 mg/kg rhASM (Treatment Phase).

rhASM was intravenously administered via a bolus injection in a lateraltail vein to male and female ASMKO mice. Groups 1-3 received 3 mg/kgrhASM during the Debulking Phase (SD 1, 3, 5 and 7). Groups 1, 2, and 3received 3, 10 and 30 mg/kg rhASM, respectively, during the TreatmentPhase (SD 9, 23, 37, 51, 65, 79, and 93). One subset of mice in eachgroup (the first 2 mice/sex in Group 1 and the first 4 mice/sex inGroups 2-3) were bled pre-study as well as 5 minutes and 4 hoursfollowing Treatment Phase Doses 1, 4, and 7 for analysis oftoxicokinetics (TK) (5 minutes) and ceramide levels (4 hours). The othersubset of mice in each group (the last 2 mice/sex in Group 1 and thelast 4 mice/sex in Groups 2-3) were bled pre-study as well as 4 and 24hours following Treatment Phase Doses 1, 4, and 7 for analysis of rodentmulti-analyte profiles (4 hours) and acute phase protein/liver functiontest levels (24 hours). See the Study Design in Table 2, infra.

Due to the likelihood of anaphylactic responses beginning with the firstTreatment Phase dose and each dose thereafter, each mouse was observedclosely for signs of anaphylaxis (e.g., restlessness, chewing, rubbingface, urticaria, edema, lethargy, and scruffy coat) following testarticle administration. Mice received diphenhydramine (DPH) beginningprior to the first dose of the Treatment Phase and each dose thereafter.

DPH at a concentration of 5 mg/mL was prepared by diluting a 50 mg/mLcommercially-available stock solution with sterile 0.9% saline. DPH wasadministered intraperitoneally (IP) at a dose of 20 mg/kg (4 mL/kg)based on the most recent body weight, 10-20 minutes prior to dosingrhASM to prevent possible anaphylactic reactions. If any animaldemonstrated a hypersensitivity reaction post-test articleadministration, despite DPH pre-treatment, a second dose of DPH wasadministered IP at 10 mg/kg (2 mL/kg).

All mice were euthanized via CO2 asphyxiation on SD 100. Followingeuthanasia blood were collected for clinical pathology andimmunogenicity analysis via a cardiac puncture. A necropsy was performedon all mice following blood collection. All tissues were preserved in10% neutral buffered formalin (NBF) followed by histopathologicalanalysis. A piece of the liver, spleen, kidney, and lung were preservedin 2% glutaraldehyde/2% paraformaldehyde for analysis of thesphingomyelin load. A piece of the liver, spleen, kidney, and lung werealso collected and stored at ≤−70° C. for possible future analysis.

Following necropsy all remaining tissues including the carcass wereplaced in 10% NBF.

TABLE 2 STUDY DESIGN: Dose Dose Level of Concentration Dose Volume DoseNo. of Animals rhASM of Treatment of Treatment Identification MaleFemale Debulking Phase Treatment (mg/ml) (mL/kg) 1-rhASM* 4 4 All micewere 3 mg/kg 0.39 7.7 administered 3 mg/kg beginning on rhASM on StudyDays 1, Study Day 9 3, 5, and 7 and every Doses were administered otherweek at a concentration of 0.39 thereafter for mg/mL at a volume of 7.713 weeks 2-rhASM* 8 8 mL/kg 10 mg/kg 1.3 beginning on Study Day 9 andevery other week thereafter for 13 weeks 3-rhASM* 8 8 30 mg/kg 3.9beginning on Study Day 9 and every other week thereafter for 13 weeks*Diphenhydramine will be administered to all mice prior to the 1^(st)Treatment Phase dose and each dose thereafter.

In Life Observations:

The first day of the study was considered SD1 (the first day of dosing).Animal body weights were taken once weekly during the course of thestudy beginning on SD-1 and were taken on the Monday of each weekthereafter. Cageside observations were made once daily Monday throughFriday. Any abnormalities and observations of normal were recorded.Post-dose clinical observations/scoring were made immediately prior to,10-20, and 50-70 minutes following each dose administration. Anyabnormalities and observations of normal were recorded. The attendingveterinarian and/or the study director was consulted in the event of anadverse reaction and appropriate actions were taken based on theirrecommendations.

Euthanasia:

If there was an adverse reaction that affected the health and well beingof an animal, then the animal was euthanized via CO2 asphyxiation,opened via the thoracic, abdominal, and cranial cavities and placed in10% neutral buffered formalin (NBF) for possible future analysis. If ananimal was found dead, the entire carcass was preserved in 10% NBF forpossible future analysis. At the end of the study all surviving animalswere euthanized by CO2 asphyxiation.

Sample Collection:

Dose Analysis: Approximately five-hundred microliters (500 μL) of testarticle from all dose levels was collected within 1-10 minutes followingformulation on each day of dosing and stored on dry ice until transferto a freezer set to maintain a temperature of ≤−70° C. Dose analysissamples were transferred on SD 1, 3, 5, 7, 9, 23, 37, 51, 65, 79, and 93and were stored at ≤−70° C. until analysis. Dose analysis samples weremeasured via the A280 assay.

Blood Collection: For the collection of blood for analysis of TK, rodentmulti-analyte profiles, and acute phase protein/liver function testlevels mice were anesthetized with a mixture of isoflurane and oxygen.Blood collections occurred on SD-1, 9, 51 and 93 (pre-study and the 1st,4th, and 7th treatment phase doses, respectively) according to the BloodCollection Tables (see Tables 4 and 5, infra) and the following text.

-   -   rhASM Toxicokinetics: The first 2 mice/sex in Group 1 and the        first 4 mice/sex in Groups 2-3 had a blood sample taken for        analysis of peak rhASM levels pre-study (SD-1), and 5 minutes        following the 1st, 4th, and 7th Treatment Phase doses. Blood        from all animals was collected from the retro-orbital plexus of        unconscious mice. Approximately 60 μL of whole blood was        collected into hematocrit tubes and allowed to clot at room        temperature for at least 1 minute. Serum were prepared from        these samples by centrifugation for 5 minutes at 10,000        revolutions per minute (RPM). Following centrifugation the serum        was collected and stored on dry ice until transfer to a freezer        set to maintain a temperature of ≤−70° C. Once transferred, all        samples were stored at ≤−70° C. until analysis. TK samples were        transferred on SD 1, 9, 51, and 93. TK samples were measured by        an enzyme linked immunosorbent assay (ELISA).    -   Analysis of Ceramide Levels: The first 2 mice/sex in Group 1 and        the first 4 mice/sex in Groups 2-3 had a blood sample taken for        analysis of ceramide levels pre-study (SD-1), and 4 hours        following the 1st, 4th, and 7th Treatment Phase doses. Blood        from these animals was collected from the retro-orbital plexus        of unconscious mice. Approximately 240 μL of whole blood was        collected into potassium EDTA tubes and placed on a Nutator        rocker for up to 30 minutes to prevent the formation of a clot.        Plasma was prepared from these samples by centrifugation for 5        minutes at 10,000 RPM. Following centrifugation the plasma was        collected and stored on dry ice until transfer to a freezer set        to maintain a temperature of ≤−70° C. Once transferred, all        samples were stored at ≤−70° C. until analysis by a Mass        Spectrometry Group. Ceramide samples were transferred no later        than SD 94. Ceramide samples were measured by mass spectrometry.    -   Analysis of Rodent Multi-Analyte Profiles (MAP): The last 2        mice/sex in Group 1 and the last 4 mice/sex in Groups 2-3 had a        blood sample taken for analysis of rodent multi-analyte profiles        pre-study (SD-1), and 4 hours following the 1st, 4th, and 7th        Treatment Phase doses. Blood from these animals was collected        from the retro-orbital plexus in unconscious mice. Approximately        150 μL of whole blood was collected into serum separator tubes        and allowed to clot for at least 30 minutes. Serum was prepared        from these samples by centrifugation for 5 minutes at 10,000        RPM. Following centrifugation the serum was collected and stored        on dry ice until transfer to a freezer set to maintain a        temperature of ≤−70° C. Once transferred, all samples were        stored at ≤−70° C. until shipment on no later than SD 94 for        analysis of rodent multi-analyte profiles. Samples were shipped        to Rules Based Medicine on dry ice. The Rodent Multi-Analyte        Profiles Table (see Table 3, infra) lists the analytes that were        measured.

TABLE 3 Rodent Multi-Analyte Profiles: Analytes to be TestedApolipoprotein Al Interleukin-2 MIP-3 beta C-Reactive ProteinInterleukin-3 MMP-9 CD40 Interleukin-4 MCP-1 CD40 Ligand Interleukin-5MCP-3 Endothelin-1 Interleukin-6 MCP-5 Eotaxin Interleukin-7Myeloperoxidase Epidermal Growth Factor Interleukin-10 Myoglobin FactorVII Interleukin-11 Oncostatin M Fibrinogen Interleukin-12p70 RANTESFGF-basic Interleukin-17 Serum Amyloid P FGF-9 KC/GRO alpha SGOT GCP-2Leukemia Inhibitory Factor Stem Cell Factor GM-CSF LymphotactinThrombopoietin GST-alpha M-CSF TIMP 1 Haptoglobin MDC Tissue FactorImmunoglobulin A MIP-1 alpha Tumor Necrosis Factor-alpha InducibleProtein-10 MIP-1 beta VCAM-1 Interferon-gamma MIP-1 gamma VEGFInterleukin-1 alpha MIP-2 von Willebrand Factor Interleukin-1 beta

-   -   Analysis of Acute Phase Protein/Liver Function Test Levels: The        last 2 mice/sex in Group 1 and the last 4 mice/sex in Groups 2-3        had a blood sample taken for analysis of acute phase protein        (serum amyloid-A and serum amyloid-P) and liver function        (bilirubin and alanine aminotransferase (ALT)) levels pre-study        (SD-1), and 24 hours following the 1st, 4th, and 7th Treatment        Phase doses. Blood from these animals was collected from the        retro-orbital plexus in unconscious mice. Approximately 150 μL        of whole blood was collected into serum separator tubes and        allowed to clot for at least 30 minutes. Serum was prepared from        these samples by centrifugation for 5 minutes at 10,000 RPM.        Following centrifugation the serum was collected and stored on        dry ice until transfer to a freezer set to maintain a        temperature of ≤−70° C. Once transferred, all samples were        stored at ≤−70° C. until shipment no later than SD 94 for acute        phase protein/liver function test analysis. Samples were shipped        to Analytics on dry ice.

TABLE 4 Pre-Study (SD-1) Blood Collection Table Rodent Map Pre- TKPre-Bleed Bleed Ceramide Pre-Bleed APP/Liver Function Pre-Bleed GroupAnimal # Sex (60 μL) (150 μL) (240 μL) (150 μL) 1 1, 2 M X X 5, 6 F X X3, 4 M X X 7, 8 F X X 2 9, 10, 11, 12 M X X 17, 18, 19, 20 F X X 13, 14,15, 16 M X X 21, 22, 23, 24 F X X 3 25, 26, 27, 28 M X X 33, 34, 35, 36F X X 29, 30, 31, 32 M X X 37, 38, 39, 40 F X X

TABLE 5 SD 9, 51, and 93 Blood Collection Table TK Bleed Rodent MapBleed Ceramide Bleed APP/Liver Function Bleed (60 μL) (150 μL) (240 μL)(150 μL) Group Animal # Sex 5 Minutes Post-Dose 4 Hours Post-Dose 4Hours Post-Dose 24 Hours Post-Dose 1 1, 2 M X X 5, 6 F X X 3, 4 M X X 7,8 F X X 2 9, 10, 11, 12 M X X 17, 18, 19, 20 F X X 13, 14, 15, 16 M X X21, 22, 23, 24 F X X 3 25, 26, 27, 28 M X X 33, 34, 35, 36 F X X 29, 30,31, 32 M X X

Necropsy

Terminal Blood Collection for Analysis of Clinical Pathology andImmunogenicity: Following euthanasia, all animals had a cardiac punctureperformed for the collection of whole blood (approximately ≥500 μL) fromall main study animals for clinical pathology and immunogenicityanalysis. Approximately 150 μL of the whole blood was placed inpotassium EDTA tubes, and gently inverted for analysis of hematologyparameters. Following the gentle inversion all samples were stored atroom temperature rocking on a Nutator rocker until the samples werestored at 2-10° C. until analysis. The remaining blood (from the cardiacstick following removal of 150 μL for hematology analysis) was placed ina serum separator tube, allowed to clot at room temperature for at least30 minutes, spun in a centrifuge for 5 minutes at 10,000 RPM, and theserum will be collected. Approximately 30 μL of serum was placed in aneppendorf tube for immunogenicity analysis while the remaining serum wasplaced in an eppendorf tube for clinical chemistry analysis. Theimmunogenicity samples were stored on dry ice until they were stored ina freezer set to maintain a temperature of ≤−70° C. Immunogenicitysamples were measured by an ELISA. Clinical chemistry samples wereplaced on dry ice until all samples were stored at ≤−20° C. untilanalysis. The Hematology and Clinical Chemistry Analyte Table (see Table6, infra) lists the analytes that were measured.

TABLE 6 Hematology and Clinical Chemistry Analyte Table: HematologyParameters Clinical Chemistry Parameters Leukocyte count (total alkalinephosphatase and absolute differential) erythrocyte count total bilirubin(with direct bilirubin if total bilirubin exceeds 1 mg/dL) Hemoglobinaspartate aminotransferase Hematocrit alanine aminotransferase meancorpuscular hemoglobin gamma glutamyl transferase mean corpuscularvolume urea nitrogen mean corpuscular hemoglobin Creatinineconcentration (calculated) Absolute reticulocytes total protein Plateletcount Albumin blood cell morphology globulin and A/G (albumin/ globulin)ratio (calculated) blood smear Glucose total cholesterol TriglyceridesElectrolytes (sodium, potassium, chloride) Calcium PhosphorusResults from the analysis of hematology and clinical chemistry analysesfrom all animals was interpreted by a board certified pathologist.

Tissue Collection: Following euthanasia on SD 100, all animals weresubjected to necropsy for tissue collection. The necropsy included anexamination of the external features of the carcass, external bodyorifices, the abdominal and thoracic cavities, organs, and tissues.Gross findings were recorded. The Tissue Collection Table (see Table 7,infra) lists the tissues that were collected in 10% NBF. Gross lesionswere also collected and stored in 10% NBF. The remaining carcass werepreserved in 10% NBF.

TABLE 7 Tissue Collection Table: Tissue Tissue adrenal (2) PancreasAorta pituitary gland Brain Prostate Cecum Rectum Colon salivary gland[mandibular (2)] Duodenum seminal vesicle (2) epididymus (2) sciaticnerve Esophagus skeletal muscle (quadriceps) eye with optic nerve (2)Skin femur with bone marrow (articular spinal cord (cervical, surface ofthe distal end) thoracic and lumbar) gall bladder Spleen Heart sternumwith bone marrow Ileum Stomach Jejunum testis (2) kidney (2) ThymusLiver thyroid (2) with parathyroid lung with mainstem bronchi Tonguelymph node (mandibular) Trachea lymph node (mesenteric) urinary bladdermammary gland (females) uterus with cervix ovary (2) gross lesions

Bolded Samples Will be Weighed

Organ Weights: At the time of necropsy, the above bolded organs wereweighed; paired organs were weighed together. Weights were recorded onthe organ weight form. Organ-to-body weight percentages andorgan-to-brain weight ratios were calculated based on the body weightstaken prior to necropsy.

Histopathology: Samples from all animals as well as gross lesions fromany animal on study were transferred to histology. Preserved tissueslisted above from each animal were embedded in paraffin. All tissueswere sectioned, stained with hematoxylin and eosin, and were examinedmicroscopically. All tissue sections were examined microscopically forthe evaluation of histological changes by a board certified pathologist.

Samples of the liver, spleen, kidney, and lung from all animals werecollected and fixed in 2% glutaraldehyde/2% paraformaldehyde foranalysis of sphingomyelin load. These tissues were post-fixed inpotassium dichromate/osmium tetroxide and embedded in epon. One-micronsections were stained with tannic acid and toluidine blue for lightmicroscopic examination. The sphingomyelin load in each sample wasquantitated by computer morphometry with MetaMorph software by a boardcertified pathologist.

Samples of the liver, spleen, kidney, and lung from all animals werecollected, frozen in liquid nitrogen, and stored frozen at ≤−70° C. forpossible future analysis.

Sample Analysis:

Dose analysis was measured via A280 analysis and toxicokinetic, andimmunogenicity samples were be analyzed by ELISA.

Ceramide analysis was measured via mass spectrometry.

Hematology and clinical pathology samples were measured using the SysmexXTV 2000 IV Hematology Analyzer and the Radnox Daytona ClinicalChemistry Analyzer, respectively.

Tissue samples were processed and histopathological analysis wasinterpreted by a board certified pathologist according to SOPs. Resultsfrom the clinical pathology analysis was interpreted by a boardcertified pathologist according to SOPs.

8. EXAMPLE 3: ESCALATING DOSE OF RECOMBINANT HUMAN ACID SPHINGOMYELINASE(RHASM) ENZYME REPLACEMENT THERAPY 8.1. Introduction

The protocol described below utilizes within-patient dose escalation asan option for achieving higher repeat doses of rhASM. The safety,efficacy and pharmacokinetics (PK) of rhASM infusions at doses of 0.3,0.6, and 1.0 mg/kg administered every 2 weeks (q2w) for 40 weeks, andlong term safety and efficacy of rhASM infusions are evaluated.

8.2. Materials and Methods 8.2.1. Protocol Design

The 12 NP-type B patients to be treated are of either gender. Patientswill receive at least 1 dose of 0.1 mg/kg rhASM initially. Patients thattolerate the 0.1 mg/kg rhASM infusion are then administered escalatingdoses of 0.3 mg/kg, 0.6 mg/kg, and 1 mg/kg every 2 weeks, as tolerated.Patients who tolerate 1.0 mg/kg are then stratified by spleen volume(<12 times or ≥12 times multiples of normal) and randomized to receiveone of 2 target doses: 1.0 mg/kg rhASM or 3.0 mg/kg; patients randomizedto the 3.0 mg/kg group will escalate to 2.0 mg/kg and then 3.0 mg/kg, astolerated. Patients who do not tolerate the 0.1 mg/kg dose of rhASM arereplaced. Patients who cannot tolerate escalation at higher doses willremain at their maximum tolerated dose for the duration of the 26 weekmaintenance dose period.

All patients will have doses escalated from 0.1 mg/kg to their targetdose. Dosage adjustments during the escalation period will occur atevery 2 week intervals until the target dose is reached (if tolerated).Following the initial infusion (0.1 mg/kg rhASM) as well as subsequentdoses, patient outcomes, i.e., adverse event (AE) occurrence/severityand serum bilirubin, hsCRP, and other acute phase response proteins, andceramide, will be reviewed. The following criteria will determine thenext dose to be administered during the dose escalation period:

-   -   1. Total bilirubin value ≤2.0 mg/dL or mild AE→escalate to next        dose (if applicable)    -   2. Total bilirubin value=2.1-3.0 mg/dL or moderate AE→no dosage        escalation; repeat current dose,    -   3. Total bilirubin value >3.0 mg/dL) or severe AE→decrease dose        to previously administered/tolerated.

Any serious adverse event (SAE) judged to be related to rhASM, abilirubin value that does not decrease to <2.0 mg/dL prior to the nextscheduled dose or any AE that raises concern regarding the safety ofrhASM at the administered dose may be considered adose-limiting-toxicity (DLT). If a patient experiences a DLT, subsequentdoses for the patient should be temporarily halted. Patients may bere-challenged to receive the dose that resulted in a DLT and iftolerated, treatment will proceed as originally planned.

If a patient cannot tolerate 2 doses of 0.1 mg/kg (i.e., initialinfusion and one re-challenge dose) they will not be treated withescalating doses and will be discontinued and replaced in the study.Patients who tolerate the 0.3 mg/kg dose will receive the initial rhASMinfusion (0.1 mg/kg) and, after 2 weeks, 0.3 mg/kg rhASM (provided thedose escalation criteria are met following the 0.1 mg/kg dose).Subsequent rhASM doses will be administered during the dose escalationperiod q2w for a maximum of 18 weeks. If a patient successfullyescalates to 0.3 mg/kg and subsequently meets criteria #2 or #3 (above),the patient may be re challenged twice. If re-challenge is unsuccessful(i.e., target dose cannot be reached), the patient will continue on arhASM dose of 0.1 mg/kg for the remainder of the 40-week treatmentperiod.

Patients to be treated with the 0.6 mg/kg dose regimen will receive theinitial rhASM infusion (0.1 mg/kg), followed by 1 dose of 0.3 mg/kgrhASM, followed by 0.6 mg/kg rhASM q2w for the remainder of the 40-weektreatment period (provided the previous rhASM infusions of 0.1 mg/kg and0.3 mg/kg are well tolerated). Patients to be treated with the 1.0 mg/kgregimen will receive the initial rhASM infusion (0.1 mg/kg), followed by1 dose of 0.3 mg/kg rhASM, followed by 1 dose of 0.6 mg/kg rhASM,followed by 1.0 mg/kg rhASM q2w for the remainder of the 40-weektreatment period (provided the previous rhASM infusions of 0.1 mg/kg,0.3 mg/kg, and 0.6 mg/kg are well tolerated). If a patient successfullyescalates from 0.3 to 0.6 mg/kg or from 0.6 to 1.0 mg/kg andsubsequently meets criteria #2 or #3 (above), the patient may be rechallenged twice. If re-challenge is unsuccessful (i.e., target dosecannot be reached), the patient will continue on the lower tolerateddose for the remainder of the 40-week treatment period.

Following the 40-week treatment phase, patients may be allowed tocontinue their treatment at their maintenance dose level.

8.2.2. Patients

Each patient should meet the following inclusion criteria to be treatedin accordance with these regimens:

-   -   1. Documented ASM deficiency consistent with Niemann-Pick (N-P)        disease;    -   2. Diffusing capacity (DLCO) >20% and ≤80% of the predicted        normal value;    -   3. Spleen volume ≥8 times normal (≥1.6% of body weight);    -   4. Female patients of childbearing potential must have a serum        pregnancy test negative for β-hCG, and agree to use a medically        acceptable method of birth control for the duration of the        protocol.

8.2.3. Treatment

Patients will receive a single 0.1 mg/kg rhASM IV over an approximate 35minute time period. Administration guidelines are summarized in Table 8,below. Patients that tolerate the initial 0.1 mg/kg rhASM infusion willbe dose escalated to a target treatment dose (rhASM 0.3, 0.6 or 1.0mg/kg). All patients will have doses escalated from 0.1 mg/kg to theirtarget dose. Dosage adjustments during the escalation period will occurat every 2 week intervals until the target dose is reached.

TABLE 8 Administration of rhASM Length of Target Approximate ApproximateAdministration Dose Infusion Rate Infusion Rate (approx. time (mg/kg)(mL/hr) (mg/kg/hr) in minutes) 0.1 Step 1: 20 mL/hr over 20 Step 1: 0.1mg/kg/hr over 20  35 min (+/− 5 min), if no IAR min (+/− 5 min), if noIAR Step 2: 60 mL/hr for Step 2: 0.3 mg/kg/hr for the remainder of thethe remainder of the infusion if no IAR infusion if no IAR 0.3 Step 1:17 mL/hr over 20 Step 1: 0.1 mg/kg/hr over 20  60 min (+/− 5 min), if noIAR min (+/− 5 min), if no IAR Step 2: 50 mL/hr over 20 Step 2: 0.3mg/kg/hr over 20 min (+/− 5 min), if no IAR min (+/− 5 min), if no IARStep 3: 100 mL/hr for Step 3: 0.6 mg/kg/hr for the remainder of the theremainder of the infusion if no IAR infusion if no IAR 0.6 Step 1: 17mL/hr over 20 Step 1: 0.1 mg/kg/hr over 20  80 min (+/− 5 min), if noIAR min (+/− 5 min), if no IAR Step 2: 50 mL/hr over 20 Step 2: 0.3mg/kg/hr over 20 min (+/− 5 min), if no IAR min (+/− 5 min), if no IARStep 3: 100 mL/hr over 20 Step 3: 0.6 mg/kg/hr over 20 min (+/− 5 min),if no IAR min (+/− 5 min), if no IAR Step 4: 167 mL/hr for Step 4: 1.0mg/kg/hr for the remainder of the the remainder of the infusion if noIAR infusion if no IAR 1.0 Step 1: 10 mL/hr over 20 Step 1: 0.1 mg/kg/hrover 20 100 min (+/− 5 min), if no IAR min (+/− 5 min), if no IAR Step2: 30 mL/hr over 20 Step 2: 0.3 mg/kg/hr over 20 min (+/− 5 min), if noIAR min (+/− 5 min), if no IAR Step 3: 60 mL/hr over 20 Step 3: 0.6mg/kg/hr over 20 min (+/− 5 min), if no IAR min (+/− 5 min), if no IARStep 4: 100 mL/hr for Step 4: 1.0 mg/kg/hr for the remainder of the theremainder of the infusion if no IAR infusion if no IAR

Each patient's degree of splenomegaly at baseline can be recorded inmultiples of normal (x normal) as an indicator of disease severity.Normal spleen volume is equal to 0.2% of body weight.

At each visit, patients should be evaluated for new or ongoing adverseevents (AEs).

Clinical endpoints can be measured as the % change from baseline to Week26 of the maintenance dose period. The primary efficacy endpoint is adecrease in spleen volume as measured by MRI. Secondary efficacyendpoints include a decrease in liver sphingomyelin level; an increasein exercise capacity as determined by % predicted maximum workload bycycle ergometry; increased pulmonary function as a % predicted DLco;increased lung clearance which can be determined by bronchial alveolarlavage (BAL) cell count and profile, sphingomyelin, ceramide, cytokineand chitotriosidase levels. Tertiary efficacy endpoints can include adecrease in liver volume as measured by MRI; increased pulmonaryfunction which can be determined by % predicted FVC, FEV1, TLC; improvedlung appearance determined by high resolution CT scan, chest X-ray;improved lipid profile as determined by HDL, LDL, total cholesterol,triglyceride levels, and total cholesterol: HDL cholesterol ratio;improved platelet count; hemoglobin; decreased sphingomyelin levels inskin, plasma, DBS; and improvement in other biomarkers such as CCL18,ACE.

9. EXAMPLE 4: ESCALATING DOSE OF RECOMBINANT HUMAN ACID SPHINGOMYELINASE(RHASM) ENZYME REPLACEMENT THERAPY 9.1. Introduction

The protocol described below utilizes a repeat-dose, dose comparison toevaluate the safety, efficacy and pharmacokinetics of recombinant humanacid sphingomyelinase (rhASM) in adult patients with ASMD. Theobjectives of the protocol include: (i) the evaluation of the safety ofdose escalation of rhASM; (ii) the evaluation of the safety, efficacyand pharmacokinetics of the maximum tolerated or randomized dose ofrhASM administered intravenously every 2 weeks for 26 weeks; and (iii)the evaluation of the long-term safety and efficacy of rhASM infusionsadministered intravenously every 2 weeks from week 26 until protocolcompletion (at least 182 weeks in duration).

9.2. Materials and Methods 9.2.1. Patients

Each patient should meet the following inclusion criteria to be treatedin accordance with these regimens:

-   -   1. Documented acid sphingomyelinase (ASM) deficiency consistent        with Niemann-Pick disease (NPD).    -   2. Two or more characteristic clinical features, including        thrombocytopenia, anemia, neutropenia, hepatomegaly,        splenomegaly, and pulmonary disease consistent with        non-neuronopathic NPD.    -   3. Diffusing capacity of carbon monoxide (DL_(CO)) >20% and ≥80%        of the predicted normal value;    -   4. Spleen volume ≥8 multiples of normal (MN) (i.e., ≥1.6% of        body weight). A partial splenectomy can be permitted if        performed ≥1 year from Screening/Baseline and residual spleen        volume is ≥8 MN.    -   5. Female patients of childbearing potential must have a        negative serum pregnancy test for β-human chorionic gonadotropin        (β-HCG) and agree to use a medically accepted method of        contraception for the duration of the protocol.

9.2.2. Protocol Design

Patients who can tolerate rhASM at 0.1 mg/kg can be enrolled in theprotocol. For each patient, protocol participation can consist of 3periods:

-   -   1. Screening/Baseline (−60 to −1 days).    -   2. Primary Treatment Period (approximately 32 to 46 weeks).    -   Dose Escalation (DE) Phase (Primary DE—approximately 6 to 16        weeks).        -   Upon safely escalating from 0.1 to 0.3 to 0.6 to 1.0 mg/kg            rhASM, patients can be stratified by spleen volume (<12 and            ≤12 MN) and randomized to either continue dosing at 1.0            mg/kg or continue dose escalation through 2.0 and 3.0 mg/kg            and then receive 3.0 mg/kg rhASM or the maximum tolerated            dose (Secondary DE—4 weeks) for the remainder of the            protocol.        -   Non-randomized patients can remain at their maximum            tolerated dose (<1.0 mg/kg rhASM).    -   Dose Maintenance (DM) Phase (26 weeks at maximum tolerated or        randomized dose).        -   3. Long-term Treatment Period (at least 182 weeks at maximum            tolerated or randomized dose)

The Screening/Baseline assessments can be completed at least 24 hrsprior to the initial infusion of rhASM. Enrollment into the protocol iscontingent upon the patient being able to tolerate 2 doses of rhASM at0.1 mg/kg. Initially, all eligible patients can receive a single dose of0.1 mg/kg rhASM on dose escalation (DE) Day 1. Patients who are unableto tolerate this dose are re-challenged 2 weeks later. Patients whocannot tolerate 2 doses of 0.1 mg/kg rhASM (i.e., initial dose andre-challenge dose) are discontinued and replaced, to ensure thatapproximately 12 patients (who can tolerate rhASM at 0.1 mg/kg) areenrolled.

Doses of rhASM can be escalated every 2 weeks from 0.1 to 0.3 to 0.6 to1.0 mg/kg during the Primary DE Phase. During the Primary DE Phase,patients can have rhASM doses safely escalated every 2 weeks from 0.1 to1.0 mg/kg (as indicated in FIG. 7).

During the Primary and Secondary DE Phases, the following doseescalation criteria can determine the next dose of rhASM to beadministered:

-   -   1. If total bilirubin (the highest value prior to the next        scheduled dose of rhASM and includes the pre infusion blood        draw) is 2.0 mg/dL or no/mild related adverse event        (AE)→Escalate to next dose.    -   2. If total bilirubin is >2.0 but <3.0 mg/dL or moderate related        AE →Repeat same dose.    -   If total bilirubin remains >2.0 mg/dL just prior to next        dose→Stop further dosing of patient temporarily (potential        dose-limiting toxicity [DLT]).    -   3. If total bilirubin is ≥3.0 mg/dL or severe related        AE→Decrease dose.    -   If total bilirubin remains >2.0 mg/dL just prior to next        dose→Stop further dosing of patient temporarily (potential DLT).    -   4. Patients can be re-challenged (at a particular dose level) as        many times as needed in the Primary DE Phase only (re-challenge        is not permitted in the Secondary DE Phase), before being        randomized or staying on their maximum tolerated dose by the end        of the 16-week Primary DE Phase.    -   5. Enrolled patients (i.e., those who can tolerate rhASM at 0.1        mg/kg) can not be replaced if they cannot escalate up to a dose        of 1.0 mg/kg and be randomized, or cannot tolerate their target        randomization dose (i.e., 1.0 or 3.0 mg/kg rhASM). These        patients can continue in the protocol at their maximum tolerated        dose.

If an infusion is missed, the patient can receive the same dose as theprevious infusion. If the visit window of ±5 days is exceeded, thatinfusion can not be done and the patient should have his/her nextinfusion at the scheduled time for that infusion.

For the purpose of this protocol, if any of the following criteria aremet, the results can be considered indicative of potential dose limitingtoxicity (DLT) for rhASM at a given dose:

-   -   Any serious adverse event (SAE) judged by the physician to be        related to rhASM, or    -   Total bilirubin remains >2.0 mg/dL just prior to next dose, or    -   Any adverse event (AE) that, in the opinion of the physician,        raises concern regarding the safety of rhASM at the administered        dose.

The severity of a related adverse event will be assessed by a physician.A mild related adverse event is usually transient and may require onlyminimal treatment or therapeutic intervention. A moderate relatedadverse event is usually alleviated with additional specific therapeuticintervention. This event interferes with usual activities of dailyliving, causing discomfort, but poses no significant or permanent riskof harm to the subject. A severe adverse event interrupts usualactivities of daily living, or significantly affects clinical status, ormay require intensive therapeutic intervention.

All patients who had doses escalated and safely tolerate 1.0 mg/kg rhASMby Week 16 can be stratified by spleen volume (<12 and ≥12 MN) andrandomized in a 1:1 ratio to either:

-   -   Continue receiving 1.0 mg/kg rhASM (or maximum tolerated dose)        every 2 weeks for 26 weeks or    -   Continue dose escalation (DE) through 2.0 and 3.0 mg/kg and then        receive 3.0 mg/kg rhASM (or maximum tolerated dose) every 2        weeks for 26 weeks.

During the Secondary DE Phase, patients randomized to the 3.0 mg/kggroup can have only 4 weeks to escalate their rhASM dose to 3.0 mg/kgbefore starting the 26-week DM Phase at 3.0 mg/kg (or maximum tolerateddose; see FIG. 7).

The Primary DE Phase can range from approximately 6 to 16 weeks induration for each patient. This can depend on individual patienttolerability issues, if any, encountered by the patient during doseescalation of rhASM. During the Primary DE Phase, each patient can havetheir dose of rhASM escalated every 2 weeks from 0.1 to 1.0 mg/kg (asindicated in FIG. 7).

The Primary DE Phase can be completed when a patient receives andtolerates a dose of 1.0 mg/kg rhASM or reaches Week 16, which everoccurs first. If no criteria leading to a dose re-challenge or adecrease in dose are met, a patient can receive a single infusion ofeach dose (i.e., a total of 4 infusions of rhASM), resulting in aPrimary DE Phase of 6 weeks. However, if criteria leading to dosere-challenge or decrease in dose are met the duration of the Primary DEPhase can be longer than 6 weeks, but will not exceed 16 weeks.

Patients who had doses escalated and safely tolerate 1.0 mg/kg by Week16 can be assigned to 1 of 2 target dose groups (i.e., 1.0 or 3.0 mg/kgrhASM).

Patients randomized to 3.0 mg/kg rhASM can enter the Secondary DE Phase(see FIG. 7), consisting of 2 infusions: the first infusion (i.e., 2.0mg/kg) being 2 weeks after the first tolerated dose of 1.0 mg/kg, andthe second infusion (i.e., 3.0 mg/kg or maximum tolerated dose) being 4weeks after the first tolerated dose of 1.0 mg/kg. Therefore theSecondary DE Phase can be completed 4 weeks after the first tolerateddose of 1.0 mg/kg. No re-challenge dose will be allowed. If the patientcannot tolerate the first dose at 2.0 mg/kg, the second rhASM dose canbe decreased to the maximum tolerated dose (i.e., 1.0 mg/kg) ortemporarily halted in the event of a potential dose limiting toxicity(DLT).

Patients can continue receiving rhASM at the maximum tolerated orrandomized dose administered IV every 2 weeks during the 26-week DMPhase of the Primary Treatment Period. This can include both randomizedand non-randomized patients.

As indicated in FIG. 7, patients in the 1.0 mg/kg dose group can startthe DM Phase at their first dose of 1.0 mg/kg following randomization,whereas patients in the 3.0 mg/kg group can start the DM Phase at theirthird dose of rhASM following randomization. Subsequent rhASM doses canbe administered IV every 2 weeks for 26 weeks in the DM Phase for allrandomized patients.

Non-randomized patients can start the DM Phase at their first dose ofrhASM 2 weeks after their 16-week DE Phase. These patients can continueon their maximum tolerated dose of rhASM (<1.0 mg/kg) every 2 weeks fora total of 26 weeks.

If a randomized patient experiences a moderate/severe related AE (i.e.,an infusion associated reaction (IAR)) during the infusion in the DMPhase, the patient may have the rhASM infusion stopped and restarted(i.e., interrupted) or the infusion rate slowed down. Provided that apotential DLT has not occurred, the patient can continue on this dose ofrhASM for the remainder of the DM Phase.

After all Week 26 protocol assessments have been completed, all patients(including non-randomized patients) can enter the Long-Term Treatmentperiod and continue rhASM treatment (at the rhASM dose that wasadministered during the DM Phase) IV every 2 weeks for at least 182weeks [3.5 years] in duration or until termination of the protocol. TheLong-Term Treatment Period includes the Protocol Completion (or patientwithdrawal/discontinuation) visit and a safety follow-up phone call (30to 37 days after the last dose of rhASM). Individual patient doses ofrhASM may be adjusted in the Long-Term Treatment Period based on theresults of the Primary Treatment Period analyses. Any required doseescalation can occur as previously described.

9.3. Treatment

Patients will receive intravenous infusions of rhASM at 0.1, 0.3, 0.6and 1.0 mg/kg every 2 weeks during the Primary DE Phase. Patientsrandomized to 3.0 mg/kg rhASM will receive 2.0 mg/kg prior to receiving3.0 mg/kg (or maximum tolerated dose) 2 weeks later during the SecondaryDE Phase. During the 26-week DM Phase, patients will be administered 1.0or 3.0 mg/kg (or maximum tolerated dose) rhASM every 2 weeks followingthe DE Phase. During the Long-Term Treatment Period, the rhASM dose thatwas administered during the DM Phase will be administered IV every 2weeks.

Patients will receive each intravenous infusion of rhASM over a periodof approximately 35 to 135 minutes (min) depending on the dose (seeTable 9 below). Protocol treatment (rhASM) will be infused using astandard programmable infusion pump and a 0.22 micron, lowprotein-binding, in-line filter. To calculate the dose in mg/kg, useactual weight for patients with body mass index (BMI)≤30 (weight inkg)/(height in meters); for patients with BMI >30, calculate the weightcorresponding to a BMI of 30 using the patient's height.

TABLE 9 Administration of rhASM Total Volume of 0.9% Sodium Chloride forInjection, Length of rhASM to be Approximate Approximate AdministrationDose mixed Infusion Rate Infusion Rate (approximate (mg/kg) (mL) (mL/hr)(mg/kg/hr) time [min]) 0.1  20 Step 1: 20 mL/hr over 20 Step 1: 0.1mg/kg/hr over 20  35 min (± 5 min), if no IAR min (± 5 min), if no IARStep 2: 60 mL/hr for Step 2: 0.3 mg/kg/hr for the remainder of the theremainder of the infusion if no IAR infusion if no IAR 0.3  50 Step 1:17 mL/hr over 20 Step 1: 0.1 mg/kg/hr over 20  60 min (± 5 min), if noIAR min (± 5 min), if no IAR Step 2: 50 mL/hr over 20 Step 2: 0.3mg/kg/hr over 20 min (± 5 min), if no IAR min (± 5 min), if no IAR Step3: 100 mL/hr for Step 3: 0.6 mg/kg/hr for the remainder of the theremainder of the infusion if no IAR infusion if no IAR 0.6 100 Step 1:17 mL/hr over 20 Step 1: 0.1 mg/kg/hr over 20  80 min (± 5 min), if noIAR min (± 5 min), if no IAR Step 2: 50 mL/hr over 20 Step 2: 0.3mg/kg/hr over 20 min (± 5 min), if no IAR min (± 5 min), if no IAR Step3: 100 mL/hr over 20 Step 3: 0.6 mg/kg/hr over 20 min (± 5 min), if noIAR min (± 5 min), if no IAR Step 4: 167 mL/hr for Step 4: 1.0 mg/kg/hrfor the remainder of the the remainder of the infusion if no IARinfusion if no IAR 1.0 100 Step 1: 10 mL/hr over 20 Step 1: 0.1 mg/kg/hrover 20 100 min (± 5 min), if no IAR min (± 5 min), if no IAR Step 2: 30mL/hr over 20 Step 2: 0.3 mg/kg/hr over 20 min (± 5 min), if no IAR min(± 5 min), if no IAR Step 3: 60 mL/hr over 20 Step 3: 0.6 mg/kg/hr over20 min (± 5 min), if no IAR min (± 5 min), if no IAR Step 4: 100 mL/hrfor Step 4: 1.0 mg/kg/hr for the remainder of the the remainder of theinfusion if no IAR infusion if no IAR 2.0 200 Step 1: 10 mL/hr over 20Step 1: 0.1 mg/kg/hr over 20 120 min (± 5 min), if no IAR min (± 5 min),if no IAR Step 2: 30 mL/hr over 20 Step 2: 0.3 mg/kg/hr over 20 min (± 5min), if no IAR min (± 5 min), if no IAR Step 3: 60 mL/hr over 20 Step3: 0.6 mg/kg/hr over 20 min (± 5 min), if no IAR min (± 5 min), if noIAR Step 4: 100 mL/hr over 20 Step 4: 1.0 mg/kg/hr over 20 min (± 5min), if no IAR min (± 5 min), if no IAR Step 5: 200 mL/hr for Step 5:2.0 mg/kg/hr for the remainder of the the remainder of the infusion ifno IAR infusion if no IAR 3.0 300 Step 1: 10 mL/hr over 20 Step 1: 0.1mg/kg/hr over 20 135 min (± 5 min), if no IAR min (± 5 min), if no IARStep 2: 30 mL/hr over 20 Step 2: 0.3 mg/kg/hr over 20 min (± 5 min), ifno IAR min (± 5 min), if no IAR Step 3: 60 mL/hr over 20 Step 3: 0.6mg/kg/hr over 20 min (± 5 min), if no IAR min (± 5 min), if no IAR Step4: 100 mL/hr over 20 Step 4: 1.0 mg/kg/hr over 20 min (± 5 min), if noIAR min (± 5 min), if no IAR Step 5: 200 mL/hr over 20 Step 5: 2.0mg/kg/hr over 20 min (± 5 min), if no IAR min (± 5 min), if no IAR Step6: 300 mL/hr for Step 6: 3.0 mg/kg/hr for the remainder of the theremainder of the infusion if no IAR infusion if no IAR hr = hour; IAR =Infusion-associated reaction; min = minutes

Clinical end points can be measured as the percent (%) change frombaseline in spleen volume multiples of normal (as measured by magneticresonance imaging) after 26 weeks of receiving the maximum tolerated orrandomized dose of rhASM. Secondary efficacy endpoints include livervolume (as measured by MRI); pulmonary imaging (by high resolutioncomputed tomography scan and chest X ray; pulmonary function tests,including percent predicted diffusing capacity of carbon monoxide,percent predicted forced vital capacity, forced expiratory volume in 1second, and total lung capacity; exercise capacity by cycle ergometry,including percent predicted maximum workload, peak oxygen consumption,and carbon dioxide production; physician global assessment of change;efficacy biomarkers such as serum chitotriosidase, CCL18, and ACE; andhematology parameters such as platelet count, hemoglobin level. Efficacyassessments can also include fasting lipids, coagulation studies,disease related biomarkers, health outcome measures, and patientphotographs.

Safety assessments can be performed prior to, during and/or subsequentto dose administration. Safety assessments that can be performedinclude: a physical examination with neurological assessment, vitalsigns (e.g., systolic and diastolic blood pressure, temperature, heartrate, respiratory rate and oxygen saturation), ECG, CHO with Doppler,blood chemistry (e.g., sodium, potassium, calcium, chloride, blood ureanitrogen, creatinine, uric acid, ALT, aspartate aminotransferase, totalbilirubin, lactate dehydrogenase, alkaline phosphate, total protein,albumin, glucose, cholesterol, etc.), hematology (e.g., a complete bloodcount), urinalysis, biomarker analysis for biomarkers related to cancer,hormones, cytokines (e.g., IL-8 and IL-6), cardiovascular risk, acutephase reactants and other cellular processes, serum anti-rhASM IgGconcentration, serum anti-rhASM IgE concentration, serum tryptaseactivity, and skin testing for hypersensitivity. Further,pharmacokinetics and pharmacodynamics assessments can be performed priorto, during and/or subsequent to dose administration. For example,sphingomyelin levels can be assessed in the liver and skin by biopsy,and in plasma and DBS by tandem mass spectrometry.

10. EMBODIMENTS

The invention is illustrated by the following non-limiting embodiments:

-   -   1. A method for treating a human subject having an acid        sphingomyelinase deficiency, comprising:        -   (a) a regimen for debulking accumulated sphingomyelin            substrate in the human subject comprising:            -   i) administering an initial low non-toxic dose of acid                sphingomyelinase (ASM) to the human subject;            -   ii) administering successively higher doses of ASM to                the human subject, and monitoring the subject for one or                more adverse side effects after each successive dose as                indicated by elevated bilirubin or a related adverse                event; and        -   (b) a maintenance regimen comprising administering a dose            equal to or less than the highest dose tolerated by the            subject as the maintenance dose for the subject.    -   2. An acid sphingomyelinase (ASM) for use in the treatment of an        acid sphingomyelinase deficiency in a human subject prepared to        be administered:        -   (a) in a regimen for debulking accumulated sphingomyelin            substrate comprising:            -   (i) administration of an initial low non-toxic dose of                acid sphingomyelinase (ASM);            -   (ii) administration of successively higher doses of ASM,                and monitoring the subject for one or more adverse side                effects after each successive dose as indicated by                elevated bilirubin or a related adverse event; and        -   (b) in a maintenance regimen comprising administration of a            dose equal to or less than the highest dose tolerated by the            subject as the maintenance dose for the subject.    -   3. The method according to paragraph 1 in which the ASM is        recombinant human ASM (rhASM).    -   4. The method according to paragraph 3 in which the initial dose        ranges is from 0.1 mg/kg to 1 mg/kg of rhASM.    -   5. The method according to paragraph 3 in which the initial dose        is 0.1 mg/kg to 0.5 mg/kg of rhASM.    -   6. The method according to paragraph 3, in which the initial        dose is 0.1 mg/kg of rhASM.    -   7. The method according to any one of paragraphs 1 and 3 to 6,        in which the successively higher doses are administered one,        two, three or four weeks after the previous dose.    -   8. The method according to paragraph 7 in which the successively        higher dose is administered one week after the previous dose.    -   9. The method according to paragraph 7 in which the successively        higher dose is administered two weeks after the previous dose.    -   10. The method according to any one of paragraphs 7, 8 and 9 in        which the successively higher dose is approximately 0.1 to 1.0        mg/kg higher than the previous dose.    -   11. The method according to any one of paragraphs 7, 8 and 9 in        which the successively higher dose is approximately 0.1 to 0.5        mg/kg higher than the previous dose.    -   12. The method according to any one of paragraphs 1, and 3 to        10, in which the highest dose tolerated by the human subject is        1 mg/kg to 3 mg/kg.    -   13. The method according to any one of paragraphs 1 to 11 in        which the highest dose tolerated is administered to the human        subject as the maintenance dose.    -   14. The method according to any one of paragraphs 1 to 11 in        which the maintenance dose is a therapeutically effective dose        less than the highest dose tolerated.    -   15. The method according to any one of paragraphs 1 to 13 which        further comprises monitoring the subject during the maintenance        regimen for one or more adverse side effects as indicated by        elevated bilirubin or a related adverse event; and adjusting the        maintenance dose.    -   16. The method according to any one of paragraphs 13 to 15, in        which the maintenance dose is administered to the subject every        two to four weeks.    -   17. The ASM according to paragraph 2 which is rhASM.    -   18. The rhASM according to paragraph 17, in which the initial        dose ranges is from 0.1 mg/kg to 1 mg/kg of rhASM.    -   19. The rhASM according to paragraph 17, in which the initial        dose is 0.1 mg/kg to 0.5 mg/kg of rhASM.    -   20. The rhASM according to paragraph 17, in which the initial        dose is 0.1 mg/kg of rhASM.    -   21. The ASM according to any one of paragraphs 2 and 17 to 20,        in which the successively higher doses are administered one,        two, three or four weeks after the previous dose.    -   22. The ASM according to paragraph 21 in which the successively        higher dose is administered one week after the previous dose.    -   23. The ASM according to paragraph 21 in which the successively        higher dose is administered two weeks after the previous dose.    -   24. The ASM according to any one of paragraphs 21, 22 and 23 in        which the successively higher dose is approximately 0.1 to 1.0        mg/kg higher than the previous dose.    -   25. The ASM according to any one of paragraphs 21, 22 and 23 in        which the successively higher dose is approximately 0.1 to 0.5        mg/kg higher than the previous dose.    -   26. The ASM according to any one of paragraphs 2 and 17 to 26,        in which the highest dose tolerated by the human subject is 1        mg/kg to 3 mg/kg.    -   27. The ASM according to any one of paragraphs 2 and 17 to 26 in        which the highest dose tolerated is administered to the human        subject as the maintenance dose.    -   28. The ASM according to paragraph 27 in which the maintenance        dose is a therapeutically effective dose less than the highest        dose tolerated.    -   29. The ASM according to paragraph 27 in which the maintenance        dose is administered to the subject every two to four weeks.    -   30. A method for treating a human subject having an acid        sphingomyelinase deficiency, comprising administering rhASM in        an escalating dose regimen at the following sequential doses:    -   (a) 0.1 mg/kg,    -   (b) 0.3 mg/kg, and    -   (c) 0.6 mg/kg;        wherein each dose of rhASM is administered at least twice, and        each dose is administered at two week intervals, and wherein the        subject is monitored for toxic side effects before elevating the        dose to the next level.    -   31. The method according to paragraph 30 further comprising a        sequential dose of 1 mg/kg in the escalating dose regimen.    -   32. The method according to paragraph 31 further comprising a        sequential dose of 2 mg/kg in the escalating dose regimen.    -   33. The method according to paragraph 32 further comprising a        sequential dose of 3 mg/kg in the escalating dose regimen.    -   34. A rhASM for use in the treatment of an acid sphingomyelinase        deficiency in a human subject prepared to be administered in an        escalating dose regimen at the following sequential doses:    -   (a) 0.1 mg/kg,    -   b) 0.3 mg/kg, and    -   c) 0.6 mg/kg;        wherein each dose of is administered at least twice, and each        dose is administered at two week intervals, and wherein the        subject is monitored for toxic side effects before elevating the        dose to the next level.    -   35. The rhASM according to paragraph 34 further comprising a        sequential dose of 1 mg/kg in the escalating dose regimen.    -   36. The rhASM according to paragraph 35 further comprising a        sequential dose of 2 mg/kg in the escalating dose regimen.    -   37. The rhASM according to paragraph 36 further comprising a        sequential dose of 3 mg/kg in the escalating dose regimen.    -   38. The method according to any one of paragraphs 1, 3 to 16 and        30 to 33, in which the doses are administered intravenously.    -   39. The method according to any one of paragraphs 1, 3 to 16 and        30 to 33, in which the doses are administered intradermally,        subcutaneously and intramuscularly.    -   40. The method according to any one of paragraphs 1, 3 to 16 and        30 to 33, in which the acid sphingomyelinase deficiency is        Niemann Pick Disease (NPD) type A.    -   41. The method according to any one of paragraphs 1, 3 to 16 and        30 to 33, in which the acid sphingomyelinase deficiency is NPD        type B.    -   42. The method according to any one of paragraphs 1, 3 to 16 and        30 to 33, in which the human subject has a missense mutation in        the gene encoding acid sphingomyelinase.    -   43. The method according to any one of paragraphs 1, 3 to 16 and        30 to 33, in which the mutation is L302P, H421Y and R496L.    -   44. The method according to any one of paragraphs 1, 3 to 16 and        30 to 33, in which the human subject has a mutation in the gene        encoding acid sphingomyelinase and the mutation is ΔR608.    -   45. The ASM according to any one of paragraphs 2, 17 to 29 and        34 to 37, in which the doses are administered intravenously.    -   46. The ASM according to any one of paragraphs 2, 17 to 29 and        34 to 37, in which the doses are administered subcutaneously and        intramuscularly.    -   47. The ASM according to any one of paragraphs 2, 17 to 29 and        34 to 37, in which the acid sphingomyelinase deficiency is        Niemann Pick Disease (NPD) type A.    -   48. The ASM according to any one of paragraphs 2, 17 to 29 and        34 to 37, in which the acid sphingomyelinase deficiency is NPD        type B.    -   49. The ASM according to any one of paragraphs 2, 17 to 29 and        34 to 37, in which the human subject has a missense mutation in        the gene encoding acid sphingomyelinase.    -   50. The ASM according to any one of paragraphs 2, 17 to 29 and        34 to 37, in which the mutation is L302P, H421Y and R496L.    -   51. The ASM according to any one of paragraphs 2, 17 to 29 and        34 to 37, in which the human subject has a mutation in the gene        encoding acid sphingomyelinase and the mutation is ΔR608.

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

The invention is not to be limited in scope by the specific embodimentsdescribed herein. Indeed, various modifications of the invention inaddition to those described will become apparent to those skilled in theart from the foregoing description and accompanying figures. Suchmodifications are intended to fall within the scope of the appendedclaims.

1.-19. (canceled)
 20. A method for treating an acid sphingomyelinasedeficiency (ASMD), comprising: a. administering to a human subject inneed thereof at least one initial dose of 0.025 mg/kg to 0.05 mg/kgrecombinant human acid sphingomyelinase (rhASM); and b. subsequent tothe administration of the at least one initial dose, administering anescalating dose regimen to the human subject at the following sequentialdoses: (i) 0.1 mg/kg, (ii) 0.3 mg/kg, and (iii) 0.6 mg/kg, wherein eachescalating dose is administered at least once before elevating the doseto the next level.
 21. The method of claim 20, wherein each dose isadministered two weeks after the previous dose.
 22. The method of claim20, wherein the doses are administered intravenously.
 23. The method ofclaim 20, wherein the human subject is a human child.
 24. The method ofclaim 20, wherein the ASMD is Niemann Pick Disease (NPD) type A.
 25. Themethod of claim 20, wherein the ASMD is Niemann Pick Disease (NPD) typeB.
 26. The method of claim 20 further comprising the followingsequential dose: (iv) 1 mg/kg.
 27. The method of claim 26 furthercomprising the following sequential dose: (v) 2 mg/kg.
 28. The method ofclaim 27 further comprising the following sequential dose: (vi) 3 mg/kg.29. The method of claim 28, wherein each dose is administered two weeksafter the previous dose.
 30. The method of claim 20, wherein the humansubject has a missense mutation in the gene encoding acidsphingomyelinase.
 31. The method of claim 30, wherein the mutation isL302P, H421Y, or R496L.
 32. The method of claim 20, wherein the humansubject has a mutation in the gene encoding acid sphingomyelinase andthe mutation is ΔR608.
 33. The method of claim 20, wherein the dose of0.1 mg/kg is administered once before elevating the dose to the nextlevel.
 34. The method of claim 33, wherein the dose of 0.3 mg/kg isadministered twice before elevating the dose to the next level.
 35. Themethod of claim 34, wherein the dose of 0.6 mg/kg is administered twicebefore elevating the dose to the next level.
 36. The method of claim 20,which further comprises administering a maintenance dose to the humansubject.
 37. The method of claim 36, wherein the maintenance dose isadministered every two weeks.
 38. The method of claim 36, wherein themaintenance dose is the highest dose tolerated.
 39. The method of claim36, wherein the maintenance dose is 1 mg/kg to 3 mg/kg.